Peptide vaccines for cancers expressing tumor-associated antigens

ABSTRACT

The present invention provides peptides having an amino acid sequence as set forth in SEQ ID NO: 19, 22, 30, 34, 344, 358, 41, 44, 46, 48, 78, 376, 379, 80, 100, 101, 110, 111, 387, 112, 394, 114, 116, 117, 121, 395, 133, 135, 137, 426, 143, 147, 148, 149, 150, 152, 153, 154, 156, 160, 161, 162, 163, 166, 174, 178, 186, 194, 196, 202, 210, 213, 214, 217, 223, 227, 228, 233, 254, 271, 272 or 288, as well as peptides having the above-mentioned amino acid sequences in which 1, 2, or several (e.g., up to 5) amino acids are substituted, deleted, or added, provided the peptides possess cytotoxic T cell inducibility. The present invention also provides drugs for treating or preventing a disease associated with over-expression of the CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers containing as an active ingredient one or more of these peptides. The peptides of the present invention find further utility as vaccines.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/274,373,filed May 9, 2014, which is a division of U.S. application Ser. No.13/744,354, filed Jan. 17, 2013, now U.S. Pat. No. 8,759,481, which is adivision of U.S. application Ser. No. 12/542,638, filed Aug. 17, 2009,now U.S. Pat. No. 8,383,590, which is a continuation-in-part ofPCT/JP2008/000290 (WO 2008/102557) filed Feb. 21, 2008, which claimspriority to U.S. Provisional Application Ser. No. 60/902,949, filed Feb.21, 2007. U.S. application Ser. No. 12/542,638, filed Aug. 17, 2009,also claims the benefit of priority to U.S. Provisional Application Ser.No. 61/089,973 filed Aug. 19, 2008. All of these applications areincorporated herein by reference in their entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file087331-0965171-020812US-SEQLIST.TXT, created on Nov. 21, 2015, 246,041bytes, machine format IBM-PC, MS-Windows operating system, is herebyincorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

The present invention relates to the field of biological science, morespecifically to the field of cancer therapy. In particular, the presentinvention relates to novel immunogenic peptides that serve as extremelyeffective as cancer vaccines, and drugs for treating and preventingtumors containing such peptides.

BACKGROUND ART

It has been demonstrated that CD8⁺ cytotoxic T lymphocytes (CTLs)recognize epitope peptides derived from tumor-associated antigens (TAAs)presented on MHC class I molecules, and subsequently lyse the tumorcells. Since the discovery of the MAGE family as the first example ofTAAs, many other TAAs have been discovered using immunologicalapproaches (Boon T. (1993) Int J Cancer 54: 177-80.; Boon T. et al.,(1996) J Exp Med 183: 725-9.; van der Bruggen P et al., (1991) Science254: 1643-7.; Brichard V et al., (1993) J Exp Med 178: 489-95.; KawakamiY et al., (1994) J Exp Med 180: 347-52.). Some of them are now inclinical development as targets of immunotherapy. TAAs discovered todate include MAGE (van der Bruggen P et al., (1991) Science 254:1643-7.), gp100 (Kawakami Y et al., (1994) J Exp Med 180: 347-52.), SART(Shichijo S et al., (1998) J Exp Med 187:277-88.), and NY-ESO-1 (Chen Y.T. et al., (1997) Proc. Natl. Acd. Sci. USA, 94: 1914-8.). On the otherhand, certain gene products demonstrated to be somewhat specificallyover-expressed in tumor cells have been shown to be recognized astargets for inducing cellular immune responses. Such gene productsinclude p53 (Umano Y et al., (2001) Br J Cancer, 84:1052-7.), HER2/neu(Tanaka H et al., (2001) Br J Cancer, 84: 94-9.), CEA (Nukaya I et al.,(1999) Int. J. Cancer 80, 92-7.) and the like.

Despite significant progress in basic and clinical research concerningTAAs (Rosenberg S A et al., (1998) Nature Med, 4: 321-7.; Mukherji B. etal., (1995) Proc Natl Acad Sci USA, 92: 8078-82.: Hu X et al., (1996)Cancer Res, 56: 2479-83.), only a very limited number of candidate TAAssuitable for treatment of cancers are presently available. TAAs that areabundantly expressed in cancer cells, and whose expression is restrictedto cancer cells, would be promising candidates as immunotherapeutictargets.

Both HLA-A24 and HLA-A0201 are common HLA alleles in the Japanese andCaucasian populations (Date Y et. al., (1996) Tissue Antigens 47:93-101.; Kondo A et al., (1995) J Immunol 155: 4307-12.; Kubo R T etal., (1994) J Immunol 152: 3913-24.; Imanishi et al., Proceeding of theeleventh International Histocompatibility Workshop and Conference OxfordUniversity Press, Oxford, 1065 (1992); Williams F et al., (1997) TissueAntigen 49: 129-33.). Thus, antigenic peptides of cancers presented bythese HLA alleles may find particular utility in the treatment ofcancers among Japanese and Caucasian patients. Further, it is known thatthe induction of low-affinity CTL in vitro usually results from exposureto high concentrations of peptides, generating a high level of specificpeptide/MHC complexes on antigen-presenting cells (APCs), which willeffectively activate these CTL (Alexander-Miller et al., (1996) ProcNatl Acad Sci USA 93: 4102-7.).

Recently, HLA class I-binding peptide sequence can be expected usingalgorithms (Journal of Immunological Methods, (1995), Vol. 185, pp.181-190, J. Immunol., (1994), Vol. 152, pp. 163-175, protein science,(2000), Vol. 9, pp. 1838-1846). However, it is hard to say that theexpected epitope peptide can be cut to the size and expressed on thetarget cell surface with HLA molecule and recognized by CTL. Moreover,the algorithm, for example BIMAS(http://bimas.dcrt.nih.gov/cgi-bin/molbio/kenparker comboform) (Parker KC, et al., (1994) J Immunol.; 152(1):163-75.; Kuzushima K, et al.,(2001) Blood.; 98(6):1872-81.)) can suggest the HLA molecule-bindingpeptide, but the suggested peptide is not so rigorous (Bachinsky M M,et. al., Cancer Immun. 2005 Mar. 22; 5:6.). Thus TAA screening stillremains a lot of challenges and difficulties.

Recent developments in cDNA microarray technologies have enabled theconstruction of comprehensive profiles of gene expression in malignantcells as compared to normal cells (Okabe, H. et al., (2001) Cancer Res.,61, 2129-37.; Lin Y M. et al., (2002) Oncogene, 21; 4120-8.; Hasegawa S.et al., (2002) Cancer Res 62:7012-7.). This approach enables a morethorough understanding of the complex nature of cancer cells and themechanisms of carcinogenesis and facilitates the identification of geneswhose expression is deregulated in tumors (Bienz M. et al., (2000) Cell103, 311-20.). Among the transcripts identified as up-regulated incancers, CDH3 (GenBank Accession No. NM_001793; SEQ ID Nos.1, 2), EPHA4(GenBank Accession No. L36645; SEQ ID Nos.3, 4), ECT2 (GenBank AccessionNo. AY376439; SEQ ID Nos.5, 6), HIG2 (GenBank Accession No. NM_013332;SEQ ID Nos.7, 8) INHBB (GenBank Accession No. NM_002193; SEQ ID Nos.9,435, 10, 436), KIF20A (GenBank Accession No. NM_005733; SEQ ID Nos.11,12), KNTC2 (GenBank Accession No. AF017790; SEQ ID Nos.13, 14), TTK(GenBank Accession No. NM_003318; SEQ ID Nos.15, 16) and URLC10 (GenBankAccession No. NM_017527; SEQ ID Nos.17, 18) have been recentlydiscovered. The entire contents of the references are incorporated byreference herein. These genes are of particular interest to the presentinventors, being specifically up-regulated in tumor cells of the variouscancer tissues of the cases analyzed (see below). Thus, immunogenicpeptides derived from CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTKand URLC10 may find utility in selectively killing tumor cells thatexpress such antigens. The present invention addresses these and otherneeds.

Since cytotoxic drugs, such as M-VAC, often cause severe adversereactions, it is clear that thoughtful selection of novel targetmolecules on the basis of well-characterized mechanisms of action shouldbe very helpful in the development of effective anti-cancer drugs havinga minimized risk of side effects. Toward this goal, expression profileanalyses were previously performed on various cancers and normal humantissue. Such studies led to the discovery of multiple genes that arespecifically over-expressed in cancer (Lin Y M, et al., Oncogene. 2002Jun. 13; 21:4120-8.; Kitahara O, et al., Cancer Res. 2001 May 1;61:3544-9.; Suzuki C, et al., Cancer Res. 2003 Nov. 1; 63:7038-41.;Ashida S, Cancer Res. 2004 Sep. 1; 64:5963-72.; Ochi K, et al., Int JOncol. 2004 March; 24(3):647-55.; Kaneta Y, et al., Int J Oncol. 2003September; 23:681-91.; Obama K, Hepatology. 2005 June; 41:1339-48.; KatoT, et al., Cancer Res. 2005 Jul. 1; 65:5638-46.; Kitahara O, et al.,Neoplasia. 2002 July-August; 4:295-303.; Saito-Hisaminato A et al., DNARes 2002, 9: 35-45.). Examples of such genes identified asover-expressed in various cancers include, but are not limited to, CDH3,EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and URLC10. CDH3 has beenpreviously identified as over-expressed in bladder cancer, cervicalcancer, cholangiocellular carcinoma, colorectal cancer, endometriosis,gastric cancer, diffuse-type gastric cancer, non-small cell lung cancer(NSCLC), pancreatic cancer, soft tissue tumor and testicular tumor.EPHA4 has been identified in bladder cancer, cervical cancer,cholangiocellular carcinoma, endometriosis, diffuse-type gastric cancer,ovarian cancer, pancreatic cancer, prostate cancer and soft tissuetumor. ECT2 has been identified in bladder cancer, breast cancer,cervical cancer, cholangiocellular carcinoma, chronic myeloid leukemia(CML), colorectal cancer, esophageal cancer, NSCLC, lymphoma, prostatecancer, renal carcinoma and small cell lung cancer (SCLC). HIG2 has beenidentified in renal carcinoma and SCLC. INHBB has been identified incholangiocellular carcinoma, esophageal cancer, NSCLC, renal carcinoma,SCLC and soft tissue tumor. KIF20A has been identified in bladdercancer, breast cancer, cholangiocellular carcinoma, esophageal cancer,NSCLC, pancreatic cancer, prostate cancer, renal carcinoma and SCLC.KNTC2 has been identified in bladder cancer, breast cancer, cervicalcancer, cholangiocellular carcinoma, CML, colorectal cancer, esophagealcancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreaticcancer, prostate cancer, renal carcinoma, SCLC and soft tissue tumor.TTK has been identified in bladder cancer, breast cancer, cervicalcancer, cholangiocellular carcinoma, CML, colorectal cancer, esophagealcancer, liver cancer, NSCLC, lymphoma, osteosarcoma, prostate cancer,SCLC and soft tissue tumor. URLC10 has been identified in bladdercancer, cervical cancer, cholangiocellular carcinoma, esophageal cancer,gastric cancer, NSCLC, osteosarcoma, pancreatic cancer and SCLC.

BRIEF SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of theapplicable targets of immunotherapy. Because TAAs have often noimmunogenicity, the discovery of appropriate targets is of extremeimportance. As noted above, CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A,KNTC2, TTK and URLC10 have been identified as up-regulated in variouscancers. More particularly, these genes were identified using geneexpression profiling with a genome-wide cDNA microarray. As discussedabove, expression of CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTKand URLC10 has been shown to be specifically up-regulated in varioustumor cells, from pancreatic cancer cells to renal cell carcinomas. Asdescribed in Table 1, CDH3 expression is validly elevated in 26 out of34 bladder cancer, 17 out of 19 cervical cancer, all of 19cholangiocellular carcinoma, 30 out of 34 colorectal cancer, 20 out of21 endometriosis, 13 out of 20 gastric cancer, 7 out of 8 diffuse-typegastric cancer, 36 out of 37 NSCLC, all of 16 pancreatic cancer, all of21 soft tissue tumor and all of 10 testicular tumor.

Table 1 further demonstrates that:

-   -   EPHA4 expression is validly elevated in 14 out of 34 bladder        cancer, 8 out of 14 cervical cancer, 10 out of 25        cholangiocellular carcinoma, 5 out of 15 endometriosis, 5 out of        8 diffuse-type gastric cancer, all of 5 ovarian cancer, all 14        pancreatic cancer, 20 out of 51 prostate cancer and 14 out of 23        soft tissue tumor.    -   ECT2 expression is validly elevated in 17 out of 19 bladder        cancer, 5 out of 12 breast cancer, all of 14 cervical cancer,        all of 13 cholangiocellular carcinoma, all of 5 CML, 7 out of 8        colorectal cancer, 12 out of 16 esophageal cancer, 6 out of 16        NSCLC, 8 out of 10 lymphoma, 1 out of 1 pancreatic cancer, 10        out of 13 prostate cancer, 3 out of 6 renal carcinoma and 12 out        of 13 SCLC cancer.    -   HIG2 expression is validly elevated in 19 out of 20 renal cancer        and 7 out of 9 soft tissue tumor.    -   INHBB expression is validly elevated in 10 out of 21        cholangiocellular carcinoma, all of 12 esophageal cancer, 10 out        of 13 NSCLC, 22 out of 24 renal carcinoma, 8 out of 14 SCLC        cancer and 45 out of 49 soft tissue tumor.    -   KIF20A expression is validly elevated in all of 31 bladder        cancer, 38 out of 61 breast cancer, 10 out of 11        cholangiocellular carcinoma, 7 out of 19 esophageal cancer, 21        out of 22 NSCLC, all of 6 ovarian cancer, 17 out of 36 prostate        cancer, 6 out of 11 renal carcinoma and all of 15 SCLC.    -   KNTC2 expression is validly elevated in 30 out of 32 bladder        cancer, 47 out of 56 breast cancer, all of 10 cervical cancer,        16 out of 22 cholangiocellular carcinoma, 17 out of 37 CML, 3        out of 10 colorectal cancer, 11 out of 46 esophagus cancer, 15        out of 19 NSCLC, 7 out of 8 lymphoma, 20 out of 24 osteosarcoma,        3 out of 5 ovarian cancer, all of 2 pancreatic cancer, 15 out of        37 prostate cancer, 14 out of 19 renal carcinoma, all of 15 SCLC        and 40 out of 59 soft tissue tumor.    -   TTK expression is validly elevated in all of 27 bladder cancer,        25 out of 30 breast cancer, 15 out of 16 cervical cancer, all of        10 cholangiocellular carcinoma, 5 out of 7 CML, 6 out of 10        colorectal cancer, 24 out of 44 esophageal cancer, 8 out of 15        liver cancer, all of 12 NSCLC, all of 6 lymphoma, 13 out of 16        osteoblastoma, 12 out of 17 prostate cancer, all of 15 SCLC and        16 out of 33 soft tissue tumor.    -   URLC10 expression is validly elevated in all of 29 bladder        cancer, 15 out of 16 cervical cancer, all of 7 cholangiocellular        carcinoma, 7 out of 19 esophageal cancer, all of 3 gastric        cancer, 24 out of 27 NSCLC, 15 out of 19 osteosarcoma, 4 out of        5 pancreatic cancer, 33 out of 43 soft tissue tumor.

The present invention is based, at least in part, on the identificationof specific epitope peptides of the gene products of these genes (CDH3,EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and URLC10) which possessthe ability to induce cytotoxic T lymphocytes (CTLs) specific to thecorresponding molecules. As discussed in detail below, Peripheral BloodMononuclear Cells (PBMC) of healthy donor were stimulated usingHLA-A*2402 or HLA-A*0201 binding candidate peptides derived from CDH3,EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK or URLC10. CTL clonesand/or lines were then established with specific cytotoxicity againstthe HLA-A24 or HLA-A2 (HLA-A02) positive target cells pulsed with eachof the candidate peptides. These results demonstrate that these peptidesare HLA-A24 or HLA-A2 (HLA-A02) restricted epitope peptides that caninduce potent and specific immune responses against cells expressingCDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK or URLC10.

Accordingly, the present invention provides methods for treating orpreventing a disease associated with the over-expression of CDH3, EPHA4,ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK or URLC10, e.g. cancer. Suchmethods involve the step of administering to a subject in need thereof aCDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10polypeptides of the invention. Administration of such peptide(s) resultsin the induction of anti-tumor immunity. Thus, the present inventionprovides methods for inducing anti-tumor immunity in a subject, suchmethods involving the step of administering to the subject the CDH3,EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10 polypeptides,as well as pharmaceutical compositions for treating or preventing adisease associated with the over-expression of CDH3, EPHA4, ECT2, HIG2,INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancer, that include theCDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and URLC10polypeptides. Examples of such cancers include, but are not limited to,bladder cancer, breast cancer, cervical cancer, cholangiocellularcarcinoma, CML, colorectal cancer, endometriosis, esophageal cancer,gastric cancer, diffused type gastric cancer, liver cancer, NSCLC,lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.

The present invention further provides methods for preventingpost-surgery recurrence of the disease mentioned above.

Regarding the specific aims and objectives recited above, it will beunderstood by those skilled in the art that one or more aspects of thisinvention can meet certain objectives, while one or more other aspectscan meet certain other objectives. Each objective may not apply equally,in all its respects, to every aspect of this invention. As such, theobjects herein can be viewed in the alternative with respect to any oneaspect of this invention.

Additional objects and features of the invention will become more fullyapparent when the following detailed description is read in conjunctionwith the accompanying figures and examples. However, it is to beunderstood that both the foregoing summary of the invention and thefollowing detailed description are of preferred embodiments, and notrestrictive of the invention or other alternate embodiments of theinvention. In particular, while the invention is described herein withreference to a number of specific embodiments, it will be appreciatedthat the description is illustrative of the invention and is notconstructed as limiting of the invention. Various modifications andapplications may occur to those who are skilled in the art, withoutdeparting from the spirit and the scope of the invention, as describedby the appended claims. Likewise, other objects, features, benefits andadvantages of the present invention will be apparent from this summaryand certain embodiments described below, and will be readily apparent tothose skilled in the art. Such objects, features, benefits andadvantages will be apparent from the above in conjunction with theaccompanying examples, data, figures and all reasonable inferences to bedrawn therefrom, alone or with consideration of the referencesincorporated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the briefdescription of the figures and the detailed description of the presentinvention and its preferred embodiments which follows:

FIGS. 1A-1E depict the results of the screening of epitope peptides,which, in turn, demonstrate that CDH3-A24-10-332 (SEQ ID NO: 34),CDH3-A24-10-470 (SEQ ID NO: 358), CDH3-A24-9-513 (SEQ ID NO: 19),CDH3-A24-9-406 (SEQ ID NO: 22), CDH3-A24-10-807 (SEQ ID NO: 30) andCDH3-A24-10-655 (SEQ ID NO: 344) show potent IFN-gamma production. FIG.1A depicts the example of negative peptides which could not be detectedCTL-inducing ability despite possible binding activity with HLA-A*2402.FIG. 1B depicts the CTL-inducing ability of CDH3-A24-10-332 (SEQ ID NO:34). CDH3-A24-10-332 (SEQ ID NO: 34) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line that was established from the positive well #4 shown in boxedwells, demonstrated the specific response against the target cellspulsed with the epitope peptide. FIG. 1C depicts the CTL-inducingability of CDH3-A24-10-470 (SEQ ID NO: 358). CDH3-A24-10-470 (SEQ ID NO:358) demonstrated potent IFN-gamma production as compared to the controlby IFN-gamma ELISPOT assay, and CTL line that was established from thepositive well #4 shown in boxed wells, demonstrated the specificresponse against the target cells pulsed with the epitope peptide. FIG.1D depicts the CTL-inducing ability of CDH3-A24-9-513 (SEQ ID NO: 19).CDH3-A24-9-513 (SEQ ID NO: 19) demonstrated potent IFN-gamma productionas compared to the control by IFN-gamma ELISPOT assay. The well #6 shownin boxed wells in left panel demonstrated the specific response againstthe target cells pulsed with the epitope peptide. Moreover, CTL linethat was established from the positive well #5 shown in boxed wells inmiddle panel, demonstrated the specific response against the targetcells pulsed with the epitope peptide. FIG. 1E depicts the CTL-inducingability of CDH3-A24-9-406 (SEQ ID NO: 22). CDH3-A24-9-406 (SEQ ID NO:22) demonstrated potent IFN-gamma production as compared to the controlby IFN-gamma ELISPOT assay, and CTL line that was established from thepositive well #2 shown in boxed wells, demonstrated the specificresponse against the target cells pulsed with the epitope peptide.

FIGS. 1F-1G depict the results of the screening of epitope peptides,which, in turn, demonstrate that CDH3-A24-10-332 (SEQ ID NO: 34),CDH3-A24-10-470 (SEQ ID NO: 358), CDH3-A24-9-513 (SEQ ID NO: 19),CDH3-A24-9-406 (SEQ ID NO: 22), CDH3-A24-10-807 (SEQ ID NO: 30) andCDH3-A24-10-655 (SEQ ID NO: 344) show potent IFN-gamma production. FIG.1F depicts the CTL-inducing ability of CDH3-A24-10-807 (SEQ ID NO: 30).CDH3-A24-10-807 (SEQ ID NO: 30) demonstrated potent IFN-gamma productionas compared to the control by IFN-gamma ELISPOT assay, and CTL line andthe clone were established from the positive well #5 shown in boxedwells. The established CTL clone raised against the peptide demonstratedthe specific CTL activity against COS7 transfected both full length ofCDH3 gene and HLA-A24 molecule (lower right graph). On the other hand,COS7 transfected full length of CDH3 but not HLA-A24 and COS7transfected HLA-A24 but not full length of CDH3 were prepared for thenegative control. The CTL clone showed high specific CTL activityagainst COS7 that transfected both CDH3 and HLA-A24. FIG. 1G depicts theCTL-inducing ability of CDH3-A24-10-655 (SEQ ID NO: 344).CDH3-A24-10-655 (SEQ ID NO: 344) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line and the clone were established from the positive well #1 shownin boxed wells. The established CTL clone raised against the peptidedemonstrated the specific CTL activity against COS7 transfected bothfull length of CDH3 gene and HLA-A24 molecule (lower right graph). Onthe other hand, COS7 transfected full length of CDH3 but not HLA-A24 andCOS7 transfected HLA-A24 but not full length of CDH3 were prepared forthe negative control. The CTL clone showed high specific CTL activityagainst COS7 that transfected both CDH3 and HLA-A24.

FIGS. 2A-H depict the results of the screening of epitope peptides,which, in turn, demonstrate that Epha4-A24-9-453 (SEQ ID NO: 41),Epha4-A24-9-5 (SEQ ID NO: 44), Epha4-A24-9-420 (SEQ ID NO: 48),Epha4-A24-9-869 (SEQ ID NO: 46), Epha4-A24-10-24 (SEQ ID NO: 78)Epha4-A02-9-501 (SEQ ID NO: 376) and Epha4-A02-9-165 (SEQ ID NO: 379)show potent IFN-gamma production. FIG. 2A depicts the example ofnegative peptides which could not be detected CTL-inducing abilitydespite possible binding activity with HLA. FIG. 2B depicts theCTL-inducing ability of Epha4-A24-9-453 (SEQ ID NO: 41). Epha4-A24-9-453(SEQ ID NO: 41) demonstrated potent IFN-gamma production as compared tothe control by IFN-gamma ELISPOT assay, and CTL line that wasestablished from the positive well #3 shown in boxed wells, demonstratedthe specific response against the target cells pulsed with the epitopepeptide. FIG. 2C depicts the CTL-inducing ability of Epha4-A24-9-5 (SEQID NO: 44). Epha4-A24-9-5 (SEQ ID NO: 44) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line, that was established from the positive well #2 shown in boxedwells, demonstrated the specific response against the target cellspulsed with the epitope peptide. FIG. 2D depicts the CTL-inducingability of Epha4-A24-9-420 (SEQ ID NO: 48). Epha4-A24-9-420 (SEQ ID NO:48) demonstrated potent IFN-gamma production as compared to the controlby IFN-gamma ELISPOT assay. The well #6 shown in boxed wells in upperpanel demonstrated the specific response against the target cells pulsedwith the epitope peptide. Moreover CTL line that was established fromthe positive well #6 shown in boxed wells in middle panel, demonstratedthe specific response against the target cells pulsed with the epitopepeptide. FIG. 2E depicts the CTL-inducing ability of Epha4-A24-9-869(SEQ ID NO: 46). Epha4-A24-9-869 (SEQ ID NO: 46) demonstrated potentIFN-gamma production as compared to the control by IFN-gamma ELISPOTassay, and CTL line that was established from the positive well #5 shownin boxed wells, demonstrated the specific response against the targetcells pulsed with the epitope peptide. FIG. 2F depicts the CTL-inducingability of Epha4-A24-10-24 (SEQ ID NO: 78). Epha4-A24-10-24 (SEQ ID NO:78) demonstrated potent IFN-gamma production as compared to the controlby IFN-gamma ELISPOT assay, and CTL line that was established from thepositive well #4 shown in boxed wells, demonstrated the specificresponse against the target cells pulsed with the epitope peptide. FIG.2G depicts the CTL-inducing ability of Epha4-A02-9-501 (SEQ ID NO: 376).Epha4-A02-9-501 (SEQ ID NO: 376) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line and clone was established from the positive well #8 shown inboxed wells. Cytotoxic activity of the established CTL line against thetarget cells pulsed with the peptide was measured by Cr-release assay(CRA) (lower graph), and the CTL line had very potent specific cytotoxicactivity against the target cells pulsed with the peptides. FIG. 2Hdepicts the CTL-inducing ability of Epha4-A02-9-165 (SEQ ID NO: 379).Epha4-A02-9-165 (SEQ ID NO: 379) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line was established from the positive well #3 shown in boxed wells.Cytotoxic activity of the established CTL line against target cellspulsed with peptide was measured by Cr-release assay (CRA) (rightgraph), and the CTL line had very potent specific cytotoxic activityagainst the target cells pulsed with the peptides.

FIGS. 3A-3D depict the results of the screening of epitope peptides,which, in turn, demonstrate that ECT2-A24-9-515 (SEQ ID NO: 80),ECT2-A24-10-40 (SEQ ID NO: 100) and ECT2-A24-10-101 (SEQ ID NO: 101)show potent IFN-gamma production. FIG. 3A depicts the example ofnegative peptides which could not be detected CTL-inducing abilitydespite possible binding activity with HLA. FIG. 3B depicts theCTL-inducing ability of ECT2-A24-9-515 (SEQ ID NO: 80). ECT2-A24-9-515(SEQ ID NO: 80) demonstrated potent IFN-gamma production as compared tothe control by IFN-gamma ELISPOT assay. The well #5 and #7 shown inboxed wells in left panel demonstrated the specific response against thetarget cells pulsed with the epitope peptide. Moreover, CTL line thatwas established from the positive well #7 shown in boxed wells in secondpanel, demonstrated the specific response against the target cellspulsed with the epitope peptide. Cytotoxic activity of the CTL lineagainst cancer cell line, TE6 endogenously expressing ECT2 and HLA-A24was measured by Cr-release assay (CRA), and the CTL clone had verypotent cytotoxic activity against TE6. On the other hand, the effectorcells did not demonstrate the cytotoxic activity of the CTL line againstcancer cell line, TE5 expressing only ECT2 was not detected. FIG. 3Cdepicts the CTL-inducing ability of ECT2-A24-10-40 (SEQ ID NO: 100).ECT2-A24-10-40 (SEQ ID NO: 100) demonstrated potent IFN-gamma productionas compared to the control by IFN-gamma ELISPOT assay, and CTL line andthe clone were established from the positive well #2 shown in boxedwells. The established CTL clone raised against the peptide demonstratedspecific CTL activity against COS7 transfected both full length of ECT2gene and HLA-A24 molecule. On the other hand, COS7 transfected fulllength of ECT2 but not HLA-A24, COS7 transfected HLA-A24 and URLC10 geneas a substitute for full length of ECT2 and COS7 transfected HLA-A24 andpulsed with ECT2-10-101 were prepared for the negative control. The CTLclone showed high specific CTL activity against COS7 that transfectedboth ECT2 and HLA-A24. “d” depicts the CTL-inducing ability ofECT2-A24-10-101 (SEQ ID NO: 101). ECT2-A24-10-101 (SEQ ID NO: 101)demonstrated potent IFN-gamma production as compared to the control byIFN-gamma ELISPOT assay, and CTL line were established from the positivewell #1 shown in boxed wells. The established CTL line raised againstthe peptide demonstrated specific CTL activity against COS7 transfectedboth full length of ECT2 gene and HLA-A24 molecule. COS7 transfectedfull length of ECT2 but not HLA-A24, COS7 transfected HLA-A24 and URLC10gene as substitute for full length of ECT2 and COS7 transfected HLA-A24and pulsed with ECT2-10-40 were prepared for the negative control. TheCTL clone showed high specific CTL activity against COS7 thattransfected both ECT2 and HLA-A24.

FIGS. 4A-4E depict the results of the screening of epitope peptides,which, in turn, demonstrate that HIG2-A24-9-19 (SEQ ID NO: 110),HIG2-A24-9-22 (SEQ ID NO: 111), HIG2-A24-9-8 (SEQ ID NO: 387),HIG2-A24-10-7 (SEQ ID NO: 112), HIG2-A24-10-18 (SEQ ID NO: 394),HIG2-A02-9-15 (SEQ ID NO: 116), HIG2-A02-9-4 (SEQ ID NO: 117) andHIG2-A02-10-8 (SEQ ID NO: 121) show potent IFN-gamma production. FIG. 4Adepicts the example of negative peptides which could not be detectedCTL-inducing ability despite possible binding activity with HLA. FIG. 4Bdepicts the CTL-inducing ability of HIG2-A24-9-19 (SEQ ID NO: 110).HIG2-A24-9-19 (SEQ ID NO: 110) demonstrated potent IFN-gamma productionas compared to the control by IFN-gamma ELISPOT assay, and CTL line,that was established from the positive well #6 shown in boxed wells,demonstrated the specific response against the target cells pulsed withthe epitope peptide. FIG. 4C depicts the CTL-inducing ability ofHIG2-A24-9-22 (SEQ ID NO: 111). HIG2-A24-9-22 (SEQ ID NO: 111)demonstrated potent IFN-gamma production as compared to the control byIFN-gamma ELISPOT assay, and CTL line and clone, that was establishedfrom the positive well #7 shown in boxed wells, demonstrated thespecific response against the target cells pulsed with the epitopepeptide. FIG. 4D depicts the CTL-inducing ability of HIG2-A24-9-8 (SEQID NO: 387). HIG2-A24-9-8 (SEQ ID NO: 387) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line and clone, that were established from the positive well #5shown in boxed wells, demonstrated the specific response against thetarget cells pulsed with the epitope peptide. FIG. 4E depicts theCTL-inducing ability of HIG2-A02-9-8 (SEQ ID NO: 114). HIG2-A02-9-8 (SEQID NO: 114) demonstrated potent IFN-gamma production as compared to thecontrol by IFN-gamma ELISPOT assay, and CTL line was established fromthe positive well #10 shown in boxed wells. The established CTL lineraised against the peptide demonstrate specific CTL activity against293T transfected both full length of HIG2 gene and HLA-A02 molecule.293T transfected full length of HIG2 but not HLA-A02, 293 Ts transfectedHLA-A02 and FoxP3 gene as substitute for full length of HIG2 and 293 Tstransfected HLA-A02 and pulsed with HIG2-9-15 were prepared for thenegative control. The CTL line showed high specific CTL activity against293T that transfected both HIG2 and HLA-A02.

FIGS. 4F-4H depict the results of the screening of epitope peptides,which, in turn, demonstrate that HIG2-A24-9-19 (SEQ ID NO: 110),HIG2-A24-9-22 (SEQ ID NO: 111), HIG2-A24-9-8 (SEQ ID NO: 387),HIG2-A24-10-7 (SEQ ID NO: 112), HIG2-A24-10-18 (SEQ ID NO: 394),HIG2-A02-9-15 (SEQ ID NO: 116), HIG2-A02-9-4 (SEQ ID NO: 117) andHIG2-A02-10-8 (SEQ ID NO: 121) show potent IFN-gamma production. FIG. 4Fdepicts the CTL-inducing ability of HIG2-A24-10-7 (SEQ ID NO: 112).HIG2-A24-10-7 (SEQ ID NO: 112) demonstrated potent IFN-gamma productionas compared to the control by IFN-gamma ELISPOT assay, and CTL lines orclone, that were established from the positive well #1 and #7 shown inboxed wells, demonstrated the specific response against the target cellspulsed with the epitope peptide. FIG. 4G depicts the CTL-inducingability of HIG2-A24-10-18 (SEQ ID NO: 394). HIG2-A24-10-18 (SEQ ID NO:394) demonstrated potent IFN-gamma production as compared to the controlby IFN-gamma ELISPOT assay, and CTL line and clone, that wereestablished from the positive well #7 shown in boxed wells, demonstratedthe specific response against the target cells pulsed with the epitopepeptide. FIG. 4H depicts the CTL-inducing ability of HIG2-A02-9-15 (SEQID NO: 116). HIG2-A02-9-15 (SEQ ID NO: 116) demonstrated potentIFN-gamma production as compared to the control by IFN-gamma ELISPOTassay, and CTL line was established from the positive well #10 shown inboxed wells. The established CTL line raised against the peptidedemonstrated specific CTL activity against COS7 transfected both fulllength of HIG2 gene and HLA-A02 molecule. COS7 transfected full lengthof HIG2 but not HLA-A02 and COS7s transfected HLA-A02 and pulsed withHIG2-9-8 peptide were prepared for the negative control. The CTL lineshowed high specific CTL activity against COS7 that transfected bothHIG2 and HLA-A02.

FIGS. 4I-4J depict the results of the screening of epitope peptides,which, in turn, demonstrate that HIG2-A24-9-19 (SEQ ID NO: 110),HIG2-A24-9-22 (SEQ ID NO: 111), HIG2-A24-9-8 (SEQ ID NO: 387),HIG2-A24-10-7 (SEQ ID NO: 112), HIG2-A24-10-18 (SEQ ID NO: 394),HIG2-A02-9-15 (SEQ ID NO: 116), HIG2-A02-9-4 (SEQ ID NO: 117) andHIG2-A02-10-8 (SEQ ID NO: 121) show potent IFN-gamma production. FIG. 4Idepicts the CTL-inducing ability of HIG2-A02-9-4 (SEQ ID NO: 117).HIG2-A02-9-4 (SEQ ID NO: 117) demonstrated potent IFN-gamma productionas compared to the control by IFN-gamma ELISPOT assay, and CTL line andclone were established from the positive well #10 shown in boxed wells.The established CTL line raised against the peptide demonstratedspecific CTL activity against COS7 transfected both full length of HIG2gene and HLA-A02 molecule (middle graph). Also, COST transfected fulllength of HIG2 but not HLA-A02, COS7s transfected HLA-A02 and TTK geneas substitute for full length of HIG2 and COS7s transfected HLA-A02 andpulsed with HIG2-9-8 were prepared for the negative control. Cytotoxicactivity of the CTL clone against 293T, transfected both full length ofHIG2 gene and HLA-A02 molecule, and cancer cell line, Caki-1endogenously expressing HIG2 and HLA-A02 was measured by Cr-releaseassay (CRA) (lower graphs), and the CTL clone had very potent cytotoxicactivity against the transfectant with both of HIG2 gene and HLA-A02,and Caki-1. On the other hand, the effector cells did not demonstratethe cytotoxic activity of the CTL line against 293T, transfected onlyHIG2 or only HLA-A02, and cancer cell line, A498 expressing only HIG2was not detected. FIG. 4J depicts the CTL-inducing ability ofHIG2-A02-10-8 (SEQ ID NO: 121). HIG2-A02-10-8 (SEQ ID NO: 121)demonstrated potent IFN-gamma production as compared to the control byIFN-gamma ELISPOT assay, and CTL line, that was established from thepositive well #9 shown in boxed wells, demonstrated the specificresponse against the target cells pulsed with the epitope peptide.

FIGS. 5A-5C depict the results of the screening of epitope peptides,which, in turn, demonstrate that INHBB-A24-9-180 (SEQ ID NO: 395),INHBB-A24-10-180 (SEQ ID NO: 133), INHBB-A24-10-305 (SEQ ID NO: 135),INHBB-A24-10-7 (SEQ ID NO: 137) and INHBB-A24-10-212 (SEQ ID NO: 426)show potent IFN-gamma production. FIG. 5A depicts the example ofnegative peptides which could not be detected CTL-inducing abilitydespite possible binding activity with HLA. FIG. 5B depicts theCTL-inducing ability of INHBB-A24-9-180 (SEQ ID NO: 395).INHBB-A24-9-180 (SEQ ID NO: 395) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line and clone was established from the positive well #7 shown inboxed wells. Cytotoxic activity of the established CTL clone againsttumor cells, Miapaca2 expressing both of INHBB and HLA-A02 was measuredby Cr-release assay (CRA), and the effector cells showed high specificcytotoxic activity against Miapaca2. On the other hand, it did not showsignificant specific cytotoxic activity against Caki-1 expressing INHBBbut not HLA-A02. FIG. 5C depicts the CTL-inducing ability ofINHBB-A24-10-180 (SEQ ID NO: 133). INHBB-A24-10-180 (SEQ ID NO: 133)demonstrated potent IFN-gamma production as compared to the control byIFN-gamma ELISPOT assay, and CTL line was established from the positivewell #3 shown in boxed wells. The established CTL line raised againstthe peptide demonstrated high specific CTL activity against 293Ttransfected both of full length of INHBB gene and HLA-A24 molecule.Also, 293T transfected full length of INHBB but not HLA-A24 and 293 Tstransfected HLA-A24 and pulsed with INHBB-10-305 peptide were preparedfor the negative control.

FIGS. 5D-5F depict the results of the screening of epitope peptides,which, in turn, demonstrate that INHBB-A24-9-180 (SEQ ID NO: 395),INHBB-A24-10-180 (SEQ ID NO: 133), INHBB-A24-10-305 (SEQ ID NO: 135),INHBB-A24-10-7 (SEQ ID NO: 137) and INHBB-A24-10-212 (SEQ ID NO: 426)show potent IFN-gamma production. FIG. 5D depicts the CTL-inducingability of INHBB-A24-10-305 (SEQ ID NO: 135). INHBB-A24-10-305 (SEQ IDNO: 135) demonstrated potent IFN-gamma production as compared to thecontrol by IFN-gamma ELISPOT assay, and CTL line and clone wereestablished from the positive well #2 shown in boxed wells. Theestablished CTL clone raised against the peptide demonstrated highspecific CTL activity against 293T transfected both full length of INHBBgene and HLA-A24 molecule. Also, 293T transfected full length of INHBBbut HLA-A24 and 293 Ts transfected HLA-A24 and pulsed with INHBB-10-180peptide were prepared for the negative control. FIG. 5E depicts theCTL-inducing ability of INHBB-A24-10-7 (SEQ ID NO: 137)). INHBB-A24-10-7(SEQ ID NO: 137) demonstrated potent IFN-gamma production as compared tothe control by IFN-gamma ELISPOT assay, and CTL lines were establishedfrom the positive well #8 shown in boxed wells in upper panel and #2shown in boxed wells in lower panel. The CTL line from #8 welldemonstrated specific CTL activity against 293T transfected both fulllength of INHBB gene and HLA-A24 molecule. Also, 293T transfected fulllength of INHBB but not HLA-A24 and 293 Ts transfected HLA-A24 andpulsed with INHBB-10-40 peptide were prepared for the negative control.FIG. 5F depicts the CTL-inducing ability of INHBB-A24-10-212 (SEQ ID NO:426). INHBB-A24-10-212 (SEQ ID NO: 426) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line, that was established from the positive well #1 shown in boxedwells, demonstrated the specific response against the target cellspulsed with the epitope peptide.

FIGS. 6A-6E depict the results of the screening of epitope peptides,which, in turn, demonstrate that KIF20A-A24-10-304 (SEQ ID NO: 186),KIF20A-A24-9-383 (SEQ ID NO: 178), KIF20A-A24-10-66 (SEQ ID NO: 194) andKIF20A-A24-9-305 (SEQ ID NO: 174) show potent IFN-gamma production. FIG.6 A depicts the example of negative peptides which could not be detectedCTL-inducing ability despite possible binding activity with HLA. FIG. 6B depicts the CTL-inducing ability of KIF20A-A24-10-304 (SEQ ID NO:186). KIF20A-A24-10-304 (SEQ ID NO: 186) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay. Thewell #5 shown in boxed wells in lower right panel demonstrated thespecific response against the target cells pulsed with the epitopepeptide. Moreover, CTL line and clone, that were established from thepositive well #5 shown in boxed wells in upper left panel, alsodemonstrated the specific response against the target cells pulsed withthe epitope peptide. The established CTL clone raised against thepeptide demonstrated specific CTL activity against 24-LCL transfectedfull length of KIF20A gene. Also, A24-LCL transfected mock vector wasprepared for the negative control. Cytotoxic activity of the CTL cloneagainst tumor cells, Miapaca2 expressing both of KIF20A and HLA-A24 wasmeasured by Cr-release assay (CRA), and the CTL clone had very potentspecific cytotoxic activity against Miapaca2 (lower right graph). On theother hand, it did not show significant specific cytotoxic activityagainst PK59 expressing KIF20A but not HLA-A24. FIG. 6C depicts theCTL-inducing ability of KIF20A-A24-9-383 (SEQ ID NO: 178).KIF20A-A24-9-383 (SEQ ID NO: 178) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay. Thewell #3 and 4 shown in boxed wells in right panel demonstrated thespecific response against the target cells pulsed with the epitopepeptide. Moreover, CTL line, that was established from the positive well#3 shown in boxed wells in left panel, also demonstrated the specificresponse against the target cells pulsed with the epitope peptide. Theestablished CTL line demonstrated high specific CTL activity againstCOS7 transfected both full length of KIF20A gene and HLA-A24 molecule.Also, COS7 transfected full length of KIF20A but not HLA-A24 and COS7stransfected HLA-A24 and pulsed with KIF20A-9-621 peptide were preparedfor the negative control.

FIGS. 6D-6E depict the results of the screening of epitope peptides,which, in turn, demonstrate that KIF20A-A24-10-304 (SEQ ID NO: 186),KIF20A-A24-9-383 (SEQ ID NO: 178), KIF20A-A24-10-66 (SEQ ID NO: 194) andKIF20A-A24-9-305 (SEQ ID NO: 174) show potent IFN-gamma production. FIG.6D depicts the CTL-inducing ability of KIF20A-A24-10-66 (SEQ ID NO:194). KIF20A-A24-10-66 (SEQ ID NO: 194) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL lines, that were established from the positive well #6 shown inboxed wells in upper left panel and #3 shown in boxed wells in lowermiddle panel demonstrated the specific response against the target cellspulsed with the epitope peptide. Moreover, CTL clone selected from CTLline from #6 well by limiting dilution demonstrated specific CTLactivity against the target cells. The established CTL clone showedspecific CTL activity against COS7 transfected both full length ofKIF20A gene and HLA-A24 molecule. Also, COS7 transfected full length ofKIF20A but not HLA-A24, COS7s transfected HLA-A24 and URLC10 gene assubstitute for full length of KIF20A and COS7 transfected HLA-A24 andpulsed with KIF20A-10-308 peptide were prepared for the negativecontrol. FIG. 6E depicts the CTL-inducing ability of KIF20A-A24-9-305(SEQ ID NO: 174). KIF20A-A24-9-305 (SEQ ID NO: 174) demonstrated potentIFN-gamma production as compared to the control by IFN-gamma ELISPOTassay, and CTL lines, that were established from the positive well #2shown in boxed wells in upper left panel and #6 shown in boxed wells inlower middle panel, demonstrated the specific response against thetarget cells pulsed with the epitope peptide. Moreover, CTL cloneselected from CTL line from #2 well by limiting dilution demonstratedspecific CTL activity against the target cells. Cytotoxic activity ofthe CTL clone against tumor cells, PK45P expressing both of KIF20A andHLA-A24 was measured by Cr-release assay (CRA), and the CTL clone hadvery potent cytotoxic activity against PK45P. On the other hand, it didnot show significant specific cytotoxic activity against PK59 expressingKIF20A but not HLA-A24.

FIGS. 7A-7E depict the results of the screening of epitope peptides,which, in turn, demonstrate that KNTC2-A24-9-309 (SEQ ID NO: 196),KNTC2-A24-9-124 (SEQ ID NO: 202), KNTC2-A24-9-154 (SEQ ID NO: 210)KNTC2-A24-9-150 (SEQ ID NO: 213), KNTC2-A24-10-452 (SEQ ID NO: 214),KNTC2-A24-10-227 (SEQ ID NO: 217) and KNTC2-A24-10-273 (SEQ ID NO: 223)show potent IFN-gamma production. FIG. 7A depicts the example ofnegative peptides which could not be detected CTL-inducing abilitydespite possible binding activity with HLA. FIG. 7B depicts theCTL-inducing ability of KNTC2-A24-9-309 (SEQ ID NO: 196).KNTC2-A24-9-309 (SEQ ID NO: 196) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line, that was established from the positive well #8 shown in boxedwells, demonstrated the specific response against the target cellspulsed with the epitope peptide. FIG. 7C depicts the CTL-inducingability of KNTC2-A24-9-124 (SEQ ID NO: 202). KNTC2-A24-9-124 (SEQ ID NO:202) demonstrated potent IFN-gamma production as compared to the controlby IFN-gamma ELISPOT assay, and CTL line, that was established from thepositive well #5 shown in boxed wells, demonstrated the specificresponse against the target cells pulsed with the epitope peptide. FIG.7D depicts the CTL-inducing ability of KNTC2-A24-9-154 (SEQ ID NO: 210).KNTC2-A24-9-154 (SEQ ID NO: 210) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line and clone, that were established from the positive well #5shown in boxed wells demonstrated the specific response against thetarget cells pulsed with the epitope peptide. FIG. 7E depicts theCTL-inducing ability of KNTC2-A24-9-150 (SEQ ID NO: 213).KNTC2-A24-9-150 (SEQ ID NO: 213) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line, that was established from the positive well #7 shown in boxedwells, demonstrated the specific response against the target cellspulsed with the epitope peptide.

FIGS. 7F-7H depict the results of the screening of epitope peptides,which, in turn, demonstrate that KNTC2-A24-9-309 (SEQ ID NO: 196),KNTC2-A24-9-124 (SEQ ID NO: 202), KNTC2-A24-9-154 (SEQ ID NO: 210)KNTC2-A24-9-150 (SEQ ID NO: 213), KNTC2-A24-10-452 (SEQ ID NO: 214),KNTC2-A24-10-227 (SEQ ID NO: 217) and KNTC2-A24-10-273 (SEQ ID NO: 223)show potent IFN-gamma production. FIG. 7F depicts the CTL-inducingability of KNTC2-A24-10-452 (SEQ ID NO: 214). KNTC2-A24-10-452 (SEQ IDNO: 214) demonstrated potent IFN-gamma production as compared to thecontrol by IFN-gamma ELISPOT assay, and CTL lines and clone, that wereestablished from the positive well #4 shown in boxed wells in upper leftpanel and #5 shown in boxed wells in middle panel, demonstrated thespecific response against the target cells pulsed with the epitopepeptide. Moreover, CTL clone selected from CTL line from #5 well bylimiting dilution demonstrated specific CTL activity against the targetcells. The established CTL line from #4 well showed specific CTLactivity against HEK293 transfected both full length of KNTC2 gene andHLA-A24 molecule. Also, HEK293 transfected full length of KNTC2 but notHLA-A24, HEK293 transfected HLA-A24 but full length of KNTC2 and HEK293transfected HLA-A24 pulsed with KNTC-9-309 peptide were prepared for thenegative control. FIG. 7G depicts the CTL-inducing ability ofKNTC2-A24-10-227 (SEQ ID NO: 217). KNTC2-A24-10-227 (SEQ ID NO: 217)demonstrated potent IFN-gamma production as compared to the control byIFN-gamma ELISPOT assay, and CTL line, that was established from thepositive well #1 shown in boxed well s, demonstrated the specificresponse against the target cells pulsed with the epitope peptide. FIG.7H depicts the CTL-inducing ability of KNTC2-A24-10-273 (SEQ ID NO:223). KNTC2-A24-10-273 (SEQ ID NO: 223) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line, that was established from the positive well #8 shown in boxedwells, demonstrated the specific response against the target cellspulsed with the epitope peptide.

FIGS. 8A-8C depict the results of the screening of epitope peptides,which, in turn, demonstrate that TTK-A02-9-462 (SEQ ID NO: 227),TTK-A02-9-719 (SEQ ID NO: 233), TTK-A02-9-547 (SEQ ID NO: 228) andTTK-A02-10-462 (SEQ ID NO: 254), show potent IFN-gamma production. FIG.8A depicts the example of negative peptides which could not be detectedCTL-inducing ability despite possible binding activity with HLA. FIG. 8Bdepicts the CTL-inducing ability of TTK-A02-9-462 (SEQ ID NO: 227).TTK-A02-9-462 (SEQ ID NO: 227) demonstrated potent IFN-gamma productionas compared to the control by IFN-gamma ELISPOT assay, and CTL line andtwo clones, that were established from the positive well #4 shown inboxed wells, demonstrated the specific response against the target cellspulsed with the epitope peptide. The established CTL clone showed highspecific CTL activity against COS7 transfected both full length of TTKgene and HLA-A02 molecule. Also, COS7 transfected full length of TTK butnot HLA-A02, COS7s transfected HLA-A02 but not full length of TTK andCOS7s transfected HLA-A02 pulsed with TTK-9-547 peptide were preparedfor the negative control. FIG. 8C depicts the CTL-inducing ability ofTTK-A02-9-719 (SEQ ID NO: 233). TTK-A02-9-719 (SEQ ID NO: 233)demonstrated potent IFN-gamma production as compared to the control byIFN-gamma ELISPOT assay, and CTL line and clones were established fromthe positive well #1 shown in boxed wells. The established CTL lineshowed high specific CTL activity against COS7 transfected both fulllength of TTK gene and HLA-A02 molecule. Also, COS7 transfected fulllength of TTK but not HLA-A02 and COS7s transfected HLA-A02 and HIG2gene as substitute for full length of TTK were prepared for the negativecontrol.

FIG. 8D depicts the results of the screening of epitope peptides, which,in turn, demonstrate that TTK-A02-9-462 (SEQ ID NO: 227), TTK-A02-9-719(SEQ ID NO: 233), TTK-A02-9-547 (SEQ ID NO: 228) and TTK-A02-10-462 (SEQID NO: 254), show potent IFN-gamma production. FIG. 8D depicts theCTL-inducing ability of TTK-A02-9-547 (SEQ ID NO: 228). TTK-A02-9-547(SEQ ID NO: 228) demonstrated potent IFN-gamma production as compared tothe control by IFN-gamma ELISPOT assay, and CTL line and clones wereestablished from the positive well #2 shown in boxed wells. Theestablished CTL line showed specific CTL activity against COS7transfected both full length of TTK gene and HLA-A02 molecule. Also,COS7 transfected full length of TTK but not HLA-A02, COS7s transfectedHLA-A02 but not full length of TTK and COS7s transfected HLA-A02 andpulsed with TTK-10-462 were prepared for the negative control.

FIG. 8E depicts the results of the screening of epitope peptides, which,in turn, demonstrate that TTK-A02-9-462 (SEQ ID NO: 227), TTK-A02-9-719(SEQ ID NO: 233), TTK-A02-9-547 (SEQ ID NO: 228) and TTK-A02-10-462 (SEQID NO: 254), show potent IFN-gamma production. FIG. 8E depicts theCTL-inducing ability of TTK-A02-10-462 (SEQ ID NO: 254). TTK-A02-10-462(SEQ ID NO: 254) demonstrated potent IFN-gamma production as compared tothe control by IFN-gamma ELISPOT assay, and CTL line and three cloneswere established from the positive well #8 shown in boxed wells. Theestablished CTL clone showed specific CTL activity against COS7transfected both full length of TTK gene and HLA-A02 molecule. Also,COS7 transfected full length of TTK but not HLA-A02, COS7s transfectedHLA-A02 but not full length of TTK and COS7s transfected HLA-A02 andpulsed with TTK-9-547 peptide were prepared for the negative control.

FIGS. 9A-9D depict the results of the screening of epitope peptides,which, in turn, demonstrate that URLC10-A02-9-206 (SEQ ID NO: 271),URLC10-A02-9-212 (SEQ ID NO: 272) and URLC10-A02-10-211 (SEQ ID NO: 288)show potent IFN-gamma production. FIG. 9A depicts the example ofnegative peptides which could not be detected CTL-inducing abilitydespite possible binding activity with HLA. FIG. 9B depicts theCTL-inducing ability of URLC10-A02-9-206 (SEQ ID NO: 271).URLC10-A02-9-206 (SEQ ID NO: 271) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay, andCTL line, that was established from the positive well #7 shown in boxedwells, demonstrated the specific response against the target cellspulsed with the epitope peptide. FIG. 9C depicts the CTL-inducingability of URLC10-A02-9-212 (SEQ ID NO: 272). URLC10-A02-9-212 (SEQ IDNO: 272) demonstrated potent IFN-gamma production as compared to thecontrol by IFN-gamma ELISPOT assay, and CTL line, that was establishedfrom the positive well #3 shown in boxed wells, demonstrated thespecific response against the target cells pulsed with the epitopepeptide. FIG. 9D depicts the CTL-inducing ability of URLC10-A02-10-211(SEQ ID NO: 288). URLC10-A02-10-211 (SEQ ID NO: 288) demonstrated potentIFN-gamma production as compared to the control by IFN-gamma ELISPOTassay, and CTL line and clones, were established from the positive well#5 shown in boxed wells.

FIG. 9D (continuation) depicts the results of the screening of epitopepeptides, which, in turn, demonstrate that URLC10-A02-9-206 (SEQ ID NO:271), URLC10-A02-9-212 (SEQ ID NO: 272) and URLC10-A02-10-211 (SEQ IDNO: 288) show potent IFN-gamma production. FIG. 9D (continuation) Theestablished CTL clone showed high specific CTL activity against COS7,Hek293 and 293T which were transfected both full length of URLC10 geneand HLA-A02 molecule. Also, COS7, Hek293 or 293T which were transfectedfull length of URLC10 but not HLA-A02 and COS7s, Hek293s or 293 Ts,which were transfected HLA-A02 and pulsed with URLC10-10-64, wereprepared for the negative control. In this drawings, “+” means thepeptide pulsed target, “−” means the no peptide pulsed target, “R” meansResponder, “S” means Stimulator, “E” means Effector, and “T” meansTarget.

FIGS. 10A-10N include a series of photographs, (a) (n), depicting theresults of IFN-gamma ELISPOT assay on CTLs that were induced withpeptides derived from INHBB. The CTLs in well #4 stimulated withINHBB-A02-9-213 (SEQ ID NO: 143) FIG. 10A, well #5 and #7 stimulatedwith INHBB-A02-9-174 (SEQ ID NO: 147) FIG. 10B, well #8 stimulated withINHBB-A02-9-257 (SEQ ID NO: 148) FIG. 10C, well #1 and #8 stimulatedwith INHBB-A02-9-313 (SEQ ID NO: 149) FIG. 10D, well #1, #4 and #8stimulated with INHBB-A02-9-139 (SEQ ID NO: 150) FIG. 10E, well #4stimulated with INHBB-A02-9-8 (SEQ ID NO: 152) FIG. 10F, well #6stimulated with INHBB-A02-9-250 (SEQ ID NO: 153) FIG. 10G, well #5stimulated with INHBB-A02-10-179 (SEQ ID NO: 154) FIG. 10H, well #3stimulated with INHBB-A02-10-237 (SEQ ID NO: 156) FIG. 10I, well #5stimulated with INHBB-A02-10-313 (SEQ ID NO: 160) FIG. 10J, well #3 and#7 stimulated with INHBB-A02-10-173 (SEQ ID NO: 161) FIG. 10K, well #4stimulated with INHBB-A02-10-256 (SEQ ID NO: 162) FIG. 10L, well #7stimulated with INHBB-A02-10-162 (SEQ ID NO: 163) FIG. 10M and well #7stimulated with INHBB-A02-10-85 (SEQ ID NO: 166) FIG. 10N showed potentIFN-gamma production as compared with the control respectively. In thefigures, “+” indicates that the target cells in the well were pulsedwith the appropriate peptide, and “−” indicates that the target cellshad not been pulsed with any peptides.

FIG. 11 depicts a line graph showing the results of establishment of CTLlines stimulated with INHBB-A02-9-174 (SEQ ID NO: 147) with IFN-gammaELISA assay. The depicted results demonstrate that CTL line establishedby stimulation with the peptide showed potent IFN-gamma production ascompared with the control. In the figures, “+” indicates that the targetcells were pulsed with the appropriate peptide and “−” indicates thatthe target cells had not been pulsed with any peptides.

DETAILED DESCRIPTION OF THE INVENTION

The words “a”, “an”, and “the” as used herein mean “at least one” unlessotherwise specifically indicated. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

The present invention is based in part on the discovery of applicabletargets of immunotherapy. Identification of new TAAs, particularly thosethat induce potent and specific anti-tumor immune responses, warrantsfurther development of the clinical application of the peptidevaccination strategy in various types of cancer (Boon T et al., (1996) JExp Med 183: 725-9.; van der Bruggen P et al., (1991) Science 254:1643-7.; Brichard V et al., (1993) J Exp Med 178: 489-95.; Kawakami Y etal., (1994) J Exp Med 180: 347-52.; Shichijo S et al., (1998) J Exp Med187:277-88.; Chen Y T et al., (1997) Proc. Natl. Acad. Sci. USA, 94:1914-8.; Harris C C, (1996) J Natl Cancer Inst 88:1442-55.; ButterfieldL H et al., (1999) Cancer Res 59:3134-42.; Vissers J L et al., (1999)Cancer Res 59: 5554-9.; van der Burg S H et al., (1996) J. Immunol156:3308-14.; Tanaka F et al., (1997) Cancer Res 57:4465-8.; Fujie T etal., (1999) Int J Cancer 80:169-72.; Kikuchi M et al., (1999) Int JCancer 81: 459-66.; Oiso M et al., (1999) Int J Cancer 81:387-94.).Because TAAs have often no immunogenicity, discovery of fitting targetsis extremely important issue.

As noted above,

-   -   CDH3 (GenBank Accession No. NM_001793; SEQ ID Nos.1, 2),    -   EPHA4 (GenBank Accession No. L36645; SEQ ID Nos.3, 4),    -   ECT2 (GenBank Accession No. AY376439; SEQ ID Nos.5, 6),    -   HIG2 (GenBank Accession No. NM_013332; SEQ ID Nos.7, 8)    -   INHBB (GenBank Accession No. NM_002193; SEQ ID Nos.9, 435, 10,        436),    -   KIF20A (GenBank Accession No. NM_005733; SEQ ID Nos.11, 12),    -   KNTC2 (GenBank Accession No. AF017790; SEQ ID Nos.13, 14),    -   TTK (GenBank Accession No. NM_003318; SEQ ID Nos.15, 16) and    -   URLC10 (GenBank Accession No. NM_017527; SEQ ID Nos.17, 18) were        previously identified as over-expressed in various cancers using        cDNA microarray technologies.

In the present invention, peptides derived from CDH3, EPHA4, ECT2, HIG2,INHBB, KIF20A, KNTC2, TTK or URLC10 are shown to be TAA epitopesrestricted by HLA-A24 and HLA-A2 (HLA-A02), an HLA allele commonly foundin the Japanese and Caucasian populations. Specifically, using theirbinding affinities to HLA-A24 or HLA-A2 (HLA-A02), candidates of HLA-A24or HLA-A2 (HLA-A02) binding peptides derived from CDH3, EPHA4, ECT2,HIG2, INHBB, KIF20A, KNTC2, TTK or URLC10 were identified. After the invitro stimulation of T-cells by dendritic cells (DCs) loaded with thesepeptides, CTLs were successfully established using the followingpeptides.

-   -   CDH3-A24-9-513 (SEQ ID NO: 19),    -   CDH3-A24-9-406 (SEQ ID NO: 22),    -   CDH3-A24-10-807 (SEQ ID NO: 30),    -   CDH3-A24-10-332 (SEQ ID NO: 34),    -   CDH3-A24-10-655 (SEQ ID NO: 344),    -   CDH3-A24-10-470 (SEQ ID NO: 358),    -   EphA4-A24-9-453 (SEQ ID NO: 41),    -   EphA4-A24-9-5 (SEQ ID NO: 44),    -   EphA4-A24-9-869 (SEQ ID NO: 46),    -   EphA4-A24-9-420 (SEQ ID NO: 48),    -   EphA4-A24-10-24 (SEQ ID NO: 78),    -   EphA4-A02-9-501 (SEQ ID NO: 376),    -   EphA4-A02-9-165 (SEQ ID NO: 379),    -   ECT2-A24-9-515 (SEQ ID NO: 80),    -   ECT2-A24-10-40 (SEQ ID NO: 100),    -   ECT2-A24-10-101 (SEQ ID NO: 101),    -   HIG2-A24-9-19 (SEQ ID NO: 110),    -   HIG2-A24-9-22 (SEQ ID NO: 111),    -   HIG2-A24-9-8 (SEQ ID NO: 387),    -   HIG2-A24-10-7 (SEQ ID NO: 112),    -   HIG2-A24-10-18 (SEQ ID NO: 394),    -   HIG2-A02-9-8 (SEQ ID NO: 114),    -   HIG2-A02-9-15 (SEQ ID NO: 116),    -   HIG2-A02-9-4 (SEQ ID NO: 117),    -   HIG2-A02-10-8 (SEQ ID NO: 121),    -   INHBB-A24-9-180 (SEQ ID NO: 395),    -   INHBB-A24-10-180 (SEQ ID NO: 133),    -   INHBB-A24-10-305 (SEQ ID NO: 135),    -   INHBB-A24-10-7 (SEQ ID NO: 137),    -   INHBB-A24-10-212 (SEQ ID NO: 426),    -   INHBB-A02-9-213 (SEQ ID NO: 143),    -   INHBB-A02-9-174 (SEQ ID NO: 147),    -   INHBB-A02-9-257 (SEQ ID NO: 148),    -   INHBB-A02-9-313 (SEQ ID NO: 149),    -   INHBB-A02-9-139 (SEQ ID NO: 150),    -   INHBB-A02-9-8 (SEQ ID NO: 152),    -   INHBB-A02-9-250 (SEQ ID NO: 153),    -   INHBB-A02-10-179 (SEQ ID NO: 154),    -   INHBB-A02-10-237 (SEQ ID NO: 156),    -   INHBB-A02-10-313 (SEQ ID NO: 160),    -   INHBB-A02-10-173 (SEQ ID NO: 161),    -   INHBB-A02-10-256 (SEQ ID NO: 162),    -   INHBB-A02-10-162 (SEQ ID NO: 163)    -   INHBB-A02-10-85 (SEQ ID NO: 166).    -   KIF20A-A24-9-305 (SEQ ID NO: 174),    -   KIF20A-A24-9-383 (SEQ ID NO: 178),    -   KIF20A-A24-10-304 (SEQ ID NO: 186),    -   KIF20A-A24-10-66 (SEQ ID NO: 194),    -   KNTC2-A24-9-309 (SEQ ID NO: 196),    -   KNTC2-A24-9-124 (SEQ ID NO: 202),    -   KNTC2-A24-9-154 (SEQ ID NO: 210),    -   KNTC2-A24-9-150 (SEQ ID NO: 213),    -   KNTC2-A24-10-452 (SEQ ID NO: 214),    -   KNTC2-A24-10-227 (SEQ ID NO: 217),    -   KNTC2-A24-10-273 (SEQ ID NO: 223),    -   TTK-A02-9-462 (SEQ ID NO: 227),    -   TTK-A02-9-547 (SEQ ID NO: 228),    -   TTK-A02-9-719 (SEQ ID NO: 233),    -   TTK-A02-10-462 (SEQ ID NO: 254),    -   URLC-A02-9-206 (SEQ ID NO: 271),    -   URLC-A02-9-212 (SEQ ID NO: 272) and    -   URLC-A02-10-211 (SEQ ID NO: 288)

These peptides are epitope peptides of each TAA restricted by HLA-A24 orHLA-A2 (HLA-A02). Since these antigens are over-expressed in mostcancers and are associated with tumor cell proliferation, they findutility as immunotherapeutic targets against cancers. Exemplary cancersinclude, but are not limited to, bladder cancer, breast cancer, cervicalcancer, cholangiocellular carcinoma, CML, colorectal cancer,endometriosis, esophageal cancer, gastric cancer, diffused type gastriccancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer,pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissuetumor and testicular tumor.

Accordingly, the present invention further provides methods of treatingor preventing a disease associated with the over-expression of CDH3,EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancersin a subject, such methods including the steps of administering to thesubject an immunogenic peptide of less than about 40 amino acids, oftenless than about 20 amino acids, usually less than about 15 amino acidsand having the amino acid sequence of SEQ ID NOs: 19, 22, 30, 34, 344,358, 41, 44, 46, 48, 78, 376, 379, 80, 100, 101, 110, 111, 387, 112,394, 114, 116, 117, 121, 395, 133, 135, 137, 426, 143, 147, 148, 149,150, 152, 153, 154, 156, 160, 161, 162, 163, 166, 174, 178, 186, 194,196, 202, 210, 213, 214, 217, 223, 227, 228, 233, 254, 271, 272 or 288.

Alternatively, the immunogenic peptide may have an amino acid sequenceas set forth in SEQ ID NOs: 19, 22, 30, 34, 344, 358, 41, 44, 46, 48,78, 376, 379, 80, 100, 101, 110, 111, 387, 112, 394, 114, 116, 117, 121,395, 133, 135, 137, 426, 143, 147, 148, 149, 150, 152, 153, 154, 156,160, 161, 162, 163, 166, 174, 178, 186, 194, 196, 202, 210, 213, 214,217, 223, 227, 228, 233, 254, 271, 272 or 288 in which 1, 2, or several(e.g., up to 5) amino acids are substituted, deleted or added, providedthe resulting variant peptide retains the immunogenic activity (i.e.,the ability to induce CTLs specific to cells expressing CDH3, EPHA4,ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers).

The number of residues to be substituted, deleted, or added is generally5 amino acids or less, preferably 4 amino acids or less, more preferably3 amino acids or less, even more preferably one or two amino acids. Thecancers contemplated include, but are not limited to, bladder cancer,breast cancer, cervical cancer, cholangiocellular carcinoma, CML,colorectal cancer, endometriosis, esophageal cancer, gastric cancer,diffused type gastric cancer, liver cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renalcarcinoma, SCLC, soft tissue tumor and testicular tumor. Furthermore thepresent invention provides methods for preventing post-surgeryrecurrence of these diseases mentioned above.

Variant peptides (i.e., peptides having an amino acid sequence modifiedby substituting, deleting, or adding one, two or several amino acidresidues to an original amino acid sequence) are known to retain theoriginal biological activity (Mark D F et al., (1984) Proc Natl Acad SciUSA 81: 5662-6.; Zoller M J and Smith M, (1982) Nucleic Acids Res10:6487-500.; Dalbadie-McFarland G et al., (1982) Proc Natl Acad Sci USA79: 6409-13.). In the context of the present invention, it is preferablethat the amino acid modification results in conservation of theproperties of the original amino acid side-chain (a process known asconservative amino acid substitution). Examples of properties of aminoacid side chains include hydrophobic amino acids (A, I, L, M, F, P, W,Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), andside chains having the following functional groups or characteristics incommon: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl groupcontaining side-chain (S, T, Y); a sulfur atom containing side-chain (C,M); a carboxylic acid and amide containing side-chain (D, N, E, Q); abase containing side-chain (R, K, H); and an aromatic containingside-chain (H, F, Y, W). Note, the parenthetic letters indicate theone-letter codes of amino acids.

In preferred embodiments, the immunogenic peptide is a nonapeptide(9-mer) or a decapeptide (10-mer). The present invention furtherprovides a method of inducing anti-tumor immunity for a diseaseassociated with the over-expression of CDH3, EPHA4, ECT2, HIG2, INHBB,KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers, in a subject, such amethod including the steps of administering an immunogenic peptide ofthe present invention, namely one having the amino acid sequence of SEQID NOs: 19, 22, 30, 34, 344, 358, 41, 44, 46, 48, 78, 376, 379, 80, 100,101, 110, 111, 387, 112, 394, 114, 116, 117, 121, 395, 133, 135, 137,426, 143, 147, 148, 149, 150, 152, 153, 154, 156, 160, 161, 162, 163,166, 174, 178, 186, 194, 196, 202, 210, 213, 214, 217, 223, 227, 228,233, 254, 271, 272 or 288, or a variant thereof (i.e., including 1, 2,or several (e.g., up to 5) amino acid substitutions, deletions, oradditions) to the subject in need thereof. The cancers contemplatedinclude, but are not limited to, bladder cancer, breast cancer, cervicalcancer, cholangiocellular carcinoma, CML, colorectal cancer,endometriosis, esophageal cancer, gastric cancer, diffused type gastriccancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer,pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissuetumor and testicular tumor.

In the context of the present invention, the subject is preferably amammal. Exemplary mammals include, but are not limited to, e.g., ahuman, non-human primate, mouse, rat, dog, cat, horse, or cow. In thepresent invention, the peptide can be administered to a subject via anin vivo or ex vivo protocol. Furthermore, the present invention alsoprovides use of nonapeptide or decapeptide selected from peptides havingthe amino acid sequence of SEQ ID NOs: 19, 22, 30, 34, 344, 358, 41, 44,46, 48, 78, 376, 379, 80, 100, 101, 110, 111, 387, 112, 394, 114, 116,117, 121, 395, 133, 135, 137, 426, 143, 147, 148, 149, 150, 152, 153,154, 156, 160, 161, 162, 163, 166, 174, 178, 186, 194, 196, 202, 210,213, 214, 217, 223, 227, 228, 233, 254, 271, 272 or 288 (and variantsthereof) for manufacturing an immunogenic composition for treating orpreventing a disease associated with the over-expression of CDH3, EPHA4,ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers. Thecancers contemplated include, but are not limited to, bladder cancer,breast cancer, cervical cancer, cholangiocellular carcinoma, CML,colorectal cancer, endometriosis, esophageal cancer, gastric cancer,diffused type gastric cancer, liver cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renalcarcinoma, SCLC, soft tissue tumor and testicular tumor.

Homology analyses of the following peptides demonstrate that they do nothave significant homology with the peptides derived from any known humangene products.

-   -   CDH3-A24-9-513 (SEQ ID NO: 19),    -   CDH3-A24-9-406 (SEQ ID NO: 22),    -   CDH3-A24-10-807 (SEQ ID NO: 30),    -   CDH3-A24-10-332 (SEQ ID NO: 34),    -   CDH3-A24-10-655 (SEQ ID NO: 344),    -   CDH3-A24-10-470 (SEQ ID NO: 358),    -   EphA4-A24-9-453 (SEQ ID NO: 41),    -   EphA4-A24-9-5 (SEQ ID NO: 44),    -   EphA4-A24-9-869 (SEQ ID NO: 46),    -   EphA4-A24-9-420 (SEQ ID NO: 48),    -   EphA4-A24-10-24 (SEQ ID NO: 78),    -   EphA4-A02-9-501 (SEQ ID NO: 376),    -   EphA4-A02-9-165 (SEQ ID NO: 379),    -   ECT2-A24-9-515 (SEQ ID NO: 80),    -   ECT2-A24-10-40 (SEQ ID NO: 100),    -   ECT2-A24-10-101 (SEQ ID NO: 101),    -   HIG2-A24-9-19 (SEQ ID NO: 110),    -   HIG2-A24-9-22 (SEQ ID NO: 111),    -   HIG2-A24-9-8 (SEQ ID NO: 387),    -   HIG2-A24-10-7 (SEQ ID NO: 112),    -   HIG2-A24-10-18 (SEQ ID NO: 394),    -   HIG2-A02-9-8 (SEQ ID NO: 114),    -   HIG2-A02-9-15 (SEQ ID NO: 116),    -   HIG2-A02-9-4 (SEQ ID NO: 117),    -   HIG2-A02-10-8 (SEQ ID NO: 121),    -   INHBB-A24-9-180 (SEQ ID NO: 395),    -   INHBB-A24-10-180 (SEQ ID NO: 133),    -   INHBB-A24-10-305 (SEQ ID NO: 135),    -   INHBB-A24-10-7 (SEQ ID NO: 137),    -   INHBB-A24-10-212 (SEQ ID NO: 426),    -   INHBB-A02-9-213 (SEQ ID NO: 143),    -   INHBB-A02-9-174 (SEQ ID NO: 147),    -   INHBB-A02-9-257 (SEQ ID NO: 148),    -   INHBB-A02-9-313 (SEQ ID NO: 149),    -   INHBB-A02-9-139 (SEQ ID NO: 150),    -   INHBB-A02-9-8 (SEQ ID NO: 152),    -   INHBB-A02-9-250 (SEQ ID NO: 153),    -   INHBB-A02-10-179 (SEQ ID NO: 154),    -   INHBB-A02-10-237 (SEQ ID NO: 156),    -   INHBB-A02-10-313 (SEQ ID NO: 160),    -   INHBB-A02-10-173 (SEQ ID NO: 161),    -   INHBB-A02-10-256 (SEQ ID NO: 162),    -   INHBB-A02-10-162 (SEQ ID NO: 163)    -   INHBB-A02-10-85 (SEQ ID NO: 166).    -   KIF20A-A24-9-305 (SEQ ID NO: 174),    -   KIF20A-A24-9-383 (SEQ ID NO: 178),    -   KIF20A-A24-10-304 (SEQ ID NO: 186),    -   KIF20A-A24-10-66 (SEQ ID NO: 194),    -   KNTC2-A24-9-309 (SEQ ID NO: 196),    -   KNTC2-A24-9-124 (SEQ ID NO: 202),    -   KNTC2-A24-9-154 (SEQ ID NO: 210),    -   KNTC2-A24-9-150 (SEQ ID NO: 213),    -   KNTC2-A24-10-452 (SEQ ID NO: 214),    -   KNTC2-A24-10-227 (SEQ ID NO: 217),    -   KNTC2-A24-10-273 (SEQ ID NO: 223),    -   TTK-A02-9-462 (SEQ ID NO: 227),    -   TTK-A02-9-547 (SEQ ID NO: 228),    -   TTK-A02-9-719 (SEQ ID NO: 233),    -   TTK-A02-10-462 (SEQ ID NO: 254),    -   URLC-A02-9-206 (SEQ ID NO: 271),    -   URLC-A02-9-212 (SEQ ID NO: 272) and    -   URLC-A02-10-211 (SEQ ID NO: 288)

Accordingly, the possibility of unknown or undesirable immune responseswith immunotherapy against these molecules is significantly reduced.

Regarding HLA antigens, the data presented here demonstrate that theuses of A-24 type or A-2 type antigens (which are said to be highlyexpressed among the Japanese) are favorable for obtaining effectiveresults. The uses of subtypes such as A-2402 and A-0201 are even morepreferable. Typically, in the clinic, the type of HLA antigen of thepatient requiring treatment is investigated in advance, which, in turn,enables the selection of appropriate peptides having high levels ofbinding affinity to the patient antigen, or having cytotoxic T cell(CTL) inducibility by antigen presentation. Furthermore, in order toobtain peptides having high binding affinity and CTL inducibility,substitution, deletion, or addition of 1, 2, or several (e.g., up to 5)amino acids may be performed based on the amino acid sequence of thenaturally occurring CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTKand URLC10 partial peptide. Herein, the term “several” means refers to 5or less, more preferably 3 or less. Furthermore, in addition to peptidesthat are naturally displayed, since the regularity of the sequences ofpeptides displayed by binding to HLA antigens is already known (Kubo RT, et al., (1994) J. Immunol., 152, 3913-24.; Rammensee H G, et al.,(1995) Immunogenetics. 41:178-228.; Kondo A, et al., (1995) J. Immunol.155:4307-12.), modifications based on such regularity can be performedon the immunogenic peptides of the invention. For example, peptidespossessing high HLA-24 binding affinity in which the second amino acidfrom the N terminus substituted with phenylalanine, tyrosine,methionine, or tryptophan may be favorably used. Likewise, peptideswhose C-terminal amino acid is substituted with phenylalanine, leucine,isoleucine, tryptophan, or methionine may also be used favorably. On theother hand, peptides possessing high HLA-A2 (HLA-A02) binding affinityin which the second amino acid from the N terminus substituted withleucine or methionine, and peptides whose C-terminal amino acid issubstituted with valine or leucine may be used favorably. Thesubstitution is performed not only at the terminus amino acids but alsoat the position of potential TCR recognition of peptides. Severalstudies have demonstrated that amino acid substitutions in a peptide canbe equal to or better than the original, for example CAP1, p53₍₂₆₄₋₂₇₂₎, Her-2/neu ₍₃₆₉₋₃₇₇₎ or gp100 ₍₂₀₉₋₂₁₇₎ (Zaremba et al. CancerRes. 57, 4570-4577, 1997, T. K. Hoffmann et al. J Immunol. (2002) Feb.1; 168(3):1338-47., S. O. Dionne et al. Cancer Immunol Immunotherapy.(2003) 52: 199-206 and S. O. Dionne et al. Cancer Immunology,Immunotherapy (2004) 53, 307-314). Furthermore, 1 to 2 amino acids maybe added to the N terminus and/or C terminus of the peptide.

However, when the peptide sequence is identical to a portion of theamino acid sequence of an endogenous or exogenous protein having adifferent function, side effects such as autoimmune disorders orallergic symptoms against specific substances may be induced. Therefore,it is preferable to avoid the situation wherein the immunogenic sequencematches the amino acid sequence of a known protein. This situation maybe avoided by performing a homology search using available databases. Ifhomology searches confirm that peptides in which 1, 2 or severaldifferent amino acids do not exist in nature, then the danger thatmodifications of the above-mentioned amino acid sequence that, forexample, increase the binding affinity with HLA antigens, and/orincrease the CTL inducibility can be avoided.

Although peptides having high binding affinity to the HLA antigens asdescribed above are expected to be highly effective as cancer vaccines,the candidate peptides, which are selected according to the presence ofhigh binding affinity as an indicator, must be examined for the actualpresence of CTL inducibility. CTL inducibility may be routinelyconfirmed by inducing antigen-presenting cells carrying human MHCantigens (for example, B-lymphocytes, macrophages, and dendritic cells),or more specifically dendritic cells derived from human peripheral bloodmononuclear leukocytes, and, after stimulation with the peptide ofinterest, mixing with CD8-positive cells and measuring the cytotoxicactivity against the target cells. As the reaction system, transgenicanimals produced to express a human HLA antigen (for example, thosedescribed in BenMohamed L, et al., (2000) Hum. Immunol.; 61(8):764-79Related Articles, Books, Linkout.) may be used. For example, the targetcells can be radio-labeled with ⁵¹Cr and such, and cytotoxic activitycan be calculated from radioactivity released from the target cells.Alternatively, it can be examined by measuring IFN-gamma produced andreleased by CTL in the presence of antigen-presenting cells that carryimmobilized peptides, and visualizing the inhibition zone on the mediausing anti-IFN-gamma monoclonal antibodies.

As a result of examining the CTL inducibility of peptides as describedabove, it was discovered that those peptides having high bindingaffinity to an HLA antigen did not necessarily have high inducibility.However, nonapeptides or decapeptides selected from the group ofpeptides having the amino acid sequences indicated by the followingpeptides showed particularly high CTL inducibility.

-   -   CDH3-A24-9-513 (SEQ ID NO: 19),    -   CDH3-A24-9-406 (SEQ ID NO: 22),    -   CDH3-A24-10-807 (SEQ ID NO: 30),    -   CDH3-A24-10-332 (SEQ ID NO: 34),    -   CDH3-A24-10-655 (SEQ ID NO: 344),    -   CDH3-A24-10-470 (SEQ ID NO: 358),    -   EphA4-A24-9-453 (SEQ ID NO: 41),    -   EphA4-A24-9-5 (SEQ ID NO: 44),    -   EphA4-A24-9-869 (SEQ ID NO: 46),    -   EphA4-A24-9-420 (SEQ ID NO: 48),    -   EphA4-A24-10-24 (SEQ ID NO: 78),    -   EphA4-A02-9-501 (SEQ ID NO: 376),    -   EphA4-A02-9-165 (SEQ ID NO: 379),    -   ECT2-A24-9-515 (SEQ ID NO: 80),    -   ECT2-A24-10-40 (SEQ ID NO: 100),    -   ECT2-A24-10-101 (SEQ ID NO: 101),    -   HIG-A24-9-19 (SEQ ID NO: 110),    -   HIG-A24-9-22 (SEQ ID NO: 111),    -   HIG-A24-9-8 (SEQ ID NO: 387),    -   HIG-A24-10-7 (SEQ ID NO: 112),    -   HIG-A24-10-18 (SEQ ID NO: 394),    -   HIG-A02-9-8 (SEQ ID NO: 114),    -   HIG-A02-9-15 (SEQ ID NO: 116),    -   HIG-A02-9-4 (SEQ ID NO: 117),    -   HIG-A02-10-8 (SEQ ID NO: 121),    -   INHBB-A24-9-180 (SEQ ID NO: 395),    -   INHBB-A24-10-180 (SEQ ID NO: 133),    -   INHBB-A24-10-305 (SEQ ID NO: 135),    -   INHBB-A24-10-7 (SEQ ID NO: 137),    -   INHBB-A24-10-212 (SEQ ID NO: 426),    -   INHBB-A02-9-213 (SEQ ID NO: 143),    -   INHBB-A02-9-174 (SEQ ID NO: 147),    -   INHBB-A02-9-257 (SEQ ID NO: 148),    -   INHBB-A02-9-313 (SEQ ID NO: 149),    -   INHBB-A02-9-139 (SEQ ID NO: 150),    -   INHBB-A02-9-8 (SEQ ID NO: 152),    -   INHBB-A02-10-250 (SEQ ID NO: 153),    -   INHBB-A02-10-179 (SEQ ID NO: 154),    -   INHBB-A02-10-237 (SEQ ID NO: 156),    -   INHBB-A02-10-313 (SEQ ID NO: 160),    -   INHBB-A02-10-173 (SEQ ID NO: 161),    -   INHBB-A02-10-256 (SEQ ID NO: 162),    -   INHBB-A02-10-162 (SEQ ID NO: 163)    -   INHBB-A02-10-85 (SEQ ID NO: 166).    -   KIF20A-A24-9-305 (SEQ ID NO: 174),    -   KIF20A-A24-9-383 (SEQ ID NO: 178),    -   KIF20A-A24-10-304 (SEQ ID NO: 186),    -   KIF20A-A24-10-66 (SEQ ID NO: 194),    -   KNTC2-A24-9-309 (SEQ ID NO: 196),    -   KNTC2-A24-9-124 (SEQ ID NO: 202),    -   KNTC2-A24-9-154 (SEQ ID NO: 210),    -   KNTC2-A24-9-150 (SEQ ID NO: 213),    -   KNTC2-A24-10-452 (SEQ ID NO: 214),    -   KNTC2-A24-10-227 (SEQ ID NO: 217),    -   KNTC2-A24-10-273 (SEQ ID NO: 223),    -   TTK-A02-9-462 (SEQ ID NO: 227),    -   TTK-A02-9-547 (SEQ ID NO: 228),    -   TTK-A02-9-719 (SEQ ID NO: 233),    -   TTK-A02-10-462 (SEQ ID NO: 254),    -   URLC-A02-9-206 (SEQ ID NO: 271),    -   URLC-A02-9-212 (SEQ ID NO: 272) and    -   URLC-A02-10-211 (SEQ ID NO: 288)

As noted above, the present invention provides peptides having cytotoxicT cell inducibility, namely those having the amino acid sequence of SEQID NOs: 19, 22, 30, 34, 344, 358, 41, 44, 46, 48, 78, 376, 379, 80, 100,101, 110, 111, 387, 112, 394, 114, 116, 117, 121, 395, 133, 135, 137,426, 143, 147, 148, 149, 150, 152, 153, 154, 156, 160, 161, 162, 163,166, 174, 178, 186, 194, 196, 202, 210, 213, 214, 217, 223, 227, 228,233, 254, 271, 272 or 288 or a variant thereof (i.e., those in which 1,2, or several amino acids are substituted, deleted, or added).

It is preferable that the amino acid sequences composed of 9 or 10 aminoacids indicated in SEQ ID NOs: 19, 22, 30, 34, 344, 358, 41, 44, 46, 48,78, 376, 379, 80, 100, 101, 110, 111, 387, 112, 394, 114, 116, 117, 121,395, 133, 135, 137, 426, 143, 147, 148, 149, 150, 152, 153, 154, 156,160, 161, 162, 163, 166, 174, 178, 186, 194, 196, 202, 210, 213, 214,217, 223, 227, 228, 233, 254, 271, 272 or 288 or a variant thereof donot match an amino acid sequence associated with another endogenousprotein.

In particular, amino acid substitution to leucine or methionine at thesecond amino acid from the N terminus, amino acid substitution to valineor leucine at the C-terminal amino acid, and amino acid addition of 1 to2 amino acids at the N terminus and/or C terminus are examples ofpreferred variants.

One of skill in the art will recognize that in addition to amino acidsubstitutions and additions, immunologically active fragments of thepeptides may also be used in the methods of the invention. Methods fordetermining active fragments are well known in the art. CTL clonesobtained by stimulation by these modified peptides can recognize theoriginal peptides and cause damage for cells expressing the originalpeptides.

Peptides of the present invention can be prepared using well knowntechniques. For example, the peptides can be prepared synthetically,using either recombinant DNA technology or chemical synthesis. Peptidesof the present invention may be synthesized individually or as longerpolypeptides composed of two or more peptides. The peptides of thepresent invention are preferably isolated, i.e., substantially free ofother naturally occurring host cell proteins and fragments thereof.

The peptides of the present invention may contain modifications, such asglycosylation, side chain oxidation, or phosphorylation; so long as themodifications do not destroy the biological activity of the peptides asdescribed herein, namely the ability to binding to an HLA antigen andinduce CTL. Other modifications include incorporation of D-amino acidsor other amino acid mimetics that can be used, for example, to increasethe serum half life of the peptides.

Moreover, this invention may contain a method of screening for a peptidewhich 1, 2, or several amino acids are substituted, wherein said peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 19, 22, 30, 34, 344, 358, 41, 44, 46, 48, 78, 80, 100, 101,110, 111, 387, 112, 394, 395, 133, 135, 137, 426, 174, 178, 186, 194,196, 202, 210, 213, 214, 217 or 223, said method comprising the stepsof:

-   -   (a) conforming no significant sequence homology to the entire        sequence of 1, 2 or several amino acids substitute;    -   (b) measuring the CTL inducibility of the candidate substitute        peptide; and    -   (c) selecting the peptide which CTL inducibility is same to or        higher than the original peptide.        Alternatively, this invention may contain a method of screening        for a peptide which 1, 2, or several amino acids are        substituted, wherein said peptide comprises an amino acid        sequence selected from the group consisting of SEQ ID NO: 376,        379, 114, 116, 117, 121, 143, 147, 148, 149, 150, 152, 153, 154,        156, 160, 161, 162, 163, 166, 227, 228, 233, 254, 271, 272 or        288, said method comprising the steps of:    -   (a) conforming no significant sequence homology to the entire        sequence of 1, 2 or several amino acids substitute;    -   (b) measuring the CTL inducibility of the candidate substitute        peptide; and    -   (c) selecting the peptide which CTL inducibility is same to or        higher than the original peptide.

For example, in preferred embodiments, the present invention provides amethod of identifying for a peptide having an ability to induce CTLagainst cells expressing at least one tumor-associated antigen, whereinthe tumor-associated antigen is antigen selected from the groupconsisting of CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK andURLC10, said method comprising the steps of:

-   -   (i) providing or generating at least one candidate sequence        which consists of an amino acid sequence modified by        substituting, deleting, or adding one, two or several amino acid        residues to an original amino acid sequence, wherein the        original amino acid sequence is selected from the group        consisting of SEQ ID NO: 19, 22, 30, 34, 344, 358, 41, 44, 46,        48, 78, 80, 100, 101, 110, 111, 387, 112, 394, 395, 133, 135,        137, 426, 174, 178, 186, 194, 196, 202, 210, 213, 214, 217 or        223;    -   (ii) selecting the candidate sequence that does not have        substantial significant homology with the peptides derived from        any known human gene products other than said tumor-associated        antigens;    -   (iii) contacting a peptide consisting of the candidate sequence        selected in step (ii) with antigen presenting cells;    -   (iv) contacting the antigen presenting cells of step iii with        T-cells to evaluate the ability of the peptide to stimulate the        T-cells; and    -   (v) identifying the peptide of which CTL inducibility is same to        or higher than a peptide consisting of the original amino acid        sequence.        Alternatively, in preferred embodiments, the present invention        provides a method of identifying for a peptide having an ability        to induce CTL against cells expressing at least one        tumor-associated antigen, wherein the tumor-associated antigen        is antigen selected from the group consisting of CDH3, EPHA4,        ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and URLC10, said method        comprising the steps of:    -   (i) providing or generating at least one candidate sequence        which consists of an amino acid sequence modified by        substituting, deleting, or adding one, two or several amino acid        residues to an original amino acid sequence, wherein the        original amino acid sequence is selected from the group        consisting of SEQ ID NO: 376, 379, 114, 116, 117, 121, 143, 147,        148, 149, 150, 152, 153, 154, 156, 160, 161, 162, 163, 166, 227,        228, 233, 254, 271, 272 or 288;    -   (ii) selecting the candidate sequence that does not have        substantial significant homology with the peptides derived from        any known human gene products other than said tumor-associated        antigens;    -   (iii) contacting a peptide consisting of the candidate sequence        selected in step (ii) with antigen presenting cells;    -   (iv) contacting the antigen presenting cells of step (iii) with        T-cells to evaluate the ability of the peptide to stimulate the        T-cells; and    -   (v) identifying the peptide of which CTL inducibility is same to        or higher than a peptide consisting of the original amino acid        sequence.

Preferably, the amino acid is substituted for a different amino acid inwhich the properties of the amino acid side-chain are conserved (aprocess known as conservative amino acid substitution). Examples ofproperties of amino acid side chains are hydrophobic amino acids (A, I,L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H,K, S, T), and side chains having the following functional groups orcharacteristics in common: an aliphatic side-chain (G, A, V, L, I, P); ahydroxyl group containing side-chain (S, T, Y); a sulfur atom containingside-chain (C, M); a carboxylic acid and amide containing side-chain (D,N, E, Q); a base containing side-chain (R, K, H); and an aromaticcontaining side-chain (H, F, Y, W). Note, the parenthetic lettersindicate the one-letter codes of amino acids. In the present invention,substantial significant homology is, for example, more than 90%,preferably 95%, more preferably 99% or 100% identity with a known humangene product to be compared.

The peptides of this invention can be prepared as a combination, whichincludes two or more of peptides of the invention, for use as a vaccinefor a disease associated with the over-expression of CDH3, EPHA4, ECT2,HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers, such avaccine inducing CTL in vivo. The cancers contemplated include, but arenot limited to, bladder cancer, breast cancer, cervical cancer,cholangiocellular carcinoma, CML, colorectal cancer, endometriosis,esophageal cancer, gastric cancer, diffused type gastric cancer, livercancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreaticcancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor andtesticular tumor. The peptides may be in a cocktail or may be conjugatedto each other using standard techniques. For example, the peptides canbe expressed as a single polypeptide sequence. The peptides in thecombination may be the same or different.

By administering the peptides of this invention, the peptides arepresented at a high density on the HLA antigens of antigen-presentingcells, which, in turn, induces CTLs that specifically react toward thecomplex formed between the displayed peptide and the HLA antigen.Alternatively, antigen-presenting cells having immobilized the peptidesof this invention on their cell surface, obtained by removing dendriticcells from the subjects, may be stimulated by the peptides of thisinvention. Re-administration of these cells to the respective subjectsinduces CTL, and, as a result, aggressiveness towards the target cellscan be increased.

More specifically, the present invention provides drugs for treatingand/or preventing proliferation, metastasis, and such of a diseaseassociated with the over-expression of CDH3, EPHA4, ECT2, HIG2, INHBB,KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers, which include one ormore of peptides of the present invention, or a polynucleotide encodingthe peptides. The peptides or polynucleotides of the present inventionfind particular utility in the treatment of a disease associating CDH3,EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g.cancers. The cancers contemplated include, but are not limited to,bladder cancer, breast cancer, cervical cancer, cholangiocellularcarcinoma, CML, colorectal cancer, endometriosis, esophageal cancer,gastric cancer, diffused type gastric cancer, liver cancer, NSCLC,lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.

The peptides of this invention can be administered to a subjectdirectly, as a pharmaceutical composition that has been formulated byconventional formulation methods. In such cases, in addition to thepeptides of this invention, carriers, excipients, and such that areordinarily used for drugs can be included as appropriate, withoutparticular limitations. The immunogenic compositions of this inventionmay be used for treatment and prevention of a disease associated withthe over-expression of CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2,TTK and/or URLC10, e.g. cancers. The cancers contemplated include, butare not limited to, bladder cancer, breast cancer, cervical cancer,cholangiocellular carcinoma, CML, colorectal cancer, endometriosis,esophageal cancer, gastric cancer, diffused type gastric cancer, livercancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreaticcancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor andtesticular tumor.

The immunogenic compositions for treatment and/or prevention of adisease associated with the over-expression of CDH3, EPHA4, ECT2, HIG2,INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers, which include asthe active ingredient one or more peptides of the present invention, canfurther include an adjuvant so that cellular immunity will beestablished effectively. Alternatively, they may be administered withother active ingredients, such as anti-cancer agents.

The cancers contemplated include, but are not limited to, bladdercancer, breast cancer, cervical cancer, cholangiocellular carcinoma,CML, colorectal cancer, endometriosis, esophageal cancer, gastriccancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renalcarcinoma, SCLC, soft tissue tumor and testicular tumor. Suitableformulations include granules. Suitable adjuvants are described in theliterature (Johnson AG. (1994) Clin. Microbiol. Rev., 7:277-89.).

Exemplary adjuvants include, but are not limited to, aluminum phosphate,aluminum hydroxide, and alum. Furthermore, liposome formulations,granular formulations in which the drug is bound to few-mc m diameterbeads, and formulations in which a lipid is bound to the peptide may beconveniently used. The method of administration may be oral,intradermal, subcutaneous, intravenous injection, or such, and mayinclude systemic administration or local administration to the vicinityof the targeted tumor.

The dose of the peptide(s) of this invention can be adjustedappropriately according to the disease to be treated, age of thepatient, weight, method of administration, and such. Though the dosageis ordinarily 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, morepreferably 0.1 mg to 10 mg, preferably administered once in a few daysto few months, one skilled in the art can readily select the appropriatedose and method of administration, as, the selection and optimization ofthese parameters is well within routine skill.

The present invention further provides intracellular vesicles calledexosomes, which present complexes formed between the peptides of thisinvention and HLA antigens on their surface. Exosomes can be prepared,for example, by using the methods described in detail in PublishedJapanese Translation of International Publication Nos. Hei 11-510507 and2000-512161, and are preferably prepared using antigen-presenting cellsobtained from subjects who are targets of treatment and/or prevention.The exosomes of this invention can be inoculated as cancer vaccines,similarly to the peptides of this invention.

The type of HLA antigens used must match that of the subject requiringtreatment and/or prevention. For example, in the Japanese population,HLA-A24 or HLA-A2 (HLA-A02), particularly HLA-A2402 or HLA-A0201, isoften appropriate.

In some embodiments, the vaccine compositions of the present inventioninclude a component which primes cytotoxic T lymphocytes. Lipids havebeen identified as agents capable of priming CTL in vivo against viralantigens. For example, palmitic acid residues can be attached to theepsilon- and alpha-amino groups of a lysine residue and then linked toan immunogenic peptide of the invention. The lipidated peptide can thenbe administered either directly, in a micelle or particle, incorporatedinto a liposome, or emulsified in an adjuvant. As another example of alipid priming of CTL responses, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS), can be used toprime CTL when covalently attached to an appropriate peptide (see, e.g.,Deres K, et al., (1989) Nature 342:561-4.).

The immunogenic compositions of the present invention may also includenucleic acids encoding one or more of the immunogenic peptides disclosedhere. See, e.g., Wolff J A et al., (1990) Science 247:1465-8; U.S. Pat.Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;and WO 98/04720. Examples of DNA-based delivery technologies include“naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated)delivery, cationic lipid complexes, and particle-mediated (“gene gun”)or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

The immunogenic peptides of the invention can also be expressed by viralor bacterial vectors. Examples of suitable expression vectors includeattenuated viral hosts, such as vaccinia or fowlpox. This approachinvolves the use of vaccinia virus, e.g., as a vector to expressnucleotide sequences that encode the peptide. Upon introduction into ahost, the recombinant vaccinia virus expresses the immunogenic peptide,and thereby elicits an immune response. Vaccinia vectors and methodsuseful in immunization protocols are described in, e.g., U.S. Pat. No.4,722,848. Another suitable vector is BCG (Bacille Calmette Guerin). BCGvectors are described in Stover C K, et al., (1991) Nature 351:456-60. Awide variety of other vectors useful for therapeutic administration orimmunization e.g., adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, are known in the art. See, e.g., Shata M T, et al., (2000)Mol. Med. Today 6:66-71; Shedlock D J and Weiner D B., et al., (2000) J.Leukoc. Biol. 68:793-806; and Hipp J D, et al., (2000) In Vivo14:571-85.

The present invention also provides methods of inducingantigen-presenting cells using one or more peptides of this invention.The antigen-presenting cells can be induced by inducing dendritic cellsfrom the peripheral blood monocytes and then contacting (stimulating)them with one or more peptides of this invention in vitro, ex vivo or invivo. When peptides of the present invention are administered to thesubjects, antigen-presenting cells that have the peptides of thisinvention immobilized to them are induced in the body of the subject.Alternatively, after immobilizing the peptides of this invention to theantigen-presenting cells, the cells can be administered to the subjectas a vaccine. For example, the ex vivo administration may include thesteps of:

-   -   a: collecting antigen-presenting cells from a subject, and    -   b: contacting the antigen-presenting cells of step a with a        peptide of the present invention.

Alternatively, according to the present invention, use of the peptidesof this invention for manufacturing a pharmaceutical compositioninducing antigen-presenting cells is provided. Further, the presentinvention also provides the peptide of the present invention forinducing antigen-presenting cells. The antigen-presenting cells obtainedby step b can be administered to the subject as a vaccine.

This invention also provides a method for inducing antigen-presentingcells having a high level of cytotoxic T cell inducibility, in which themethod includes the step of transferring genes composed ofpolynucleotide(s) encoding one or more peptides of this invention toantigen-presenting cells in vitro. The introduced genes may be in theform of DNAs or RNAs. For the method of introduction, without particularlimitations, various methods conventionally performed in this field,such as lipofection, electroporation, and calcium phosphate method maybe suitably used. More specifically, transfection may be performed asdescribed in Reeves M E, et al., (1996) Cancer Res., 56:5672-7.;Butterfield L H, et al., (1998) J. Immunol., 161:5607-13.; Boczkowski D,et al., (1996) J. Exp. Med., 184:465-72.; Published Japanese Translationof International Publication No. 2000-509281. By transferring the geneinto antigen-presenting cells, the gene undergoes transcription,translation, and such in the cell, and then the obtained protein isprocessed by MHC Class I or Class II, and proceeds through apresentation pathway to present partial peptides.

The present invention further provides methods for inducing CTL usingone or more peptides of this invention. When the peptides of thisinvention are administered to a subject, CTL are induced in the body ofthe subject, and the strength of the immune system targeting the cellsexpressing CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/orURLC10, e.g. cancer cells in the tumor tissues is thereby enhanced.

The cancers contemplated include, but are not limited to bladder cancer,breast cancer, cervical cancer, cholangiocellular carcinoma, CML,colorectal cancer, endometriosis, esophageal cancer, gastric cancer,diffused type gastric cancer, liver cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renalcarcinoma, SCLC, soft tissue tumor and testicular tumor. Alternatively,the peptides of the present invention may be used in the context of anex vivo therapeutic method, in which subject-derived antigen-presentingcells and CD8-positive cells or peripheral blood mononuclear leukocytesare contacted (stimulated) with one or more peptides of this inventionin vitro, and, after inducing CTL, the cells are returned to thesubject. For example, the method may include the steps of:

-   -   a: collecting antigen-presenting cells from a subject,    -   b: contacting the antigen-presenting cells of step a with a        peptide of the present invention,    -   c: mixing the antigen-presenting cells of step b with CD⁸⁺ T        cells and co-culturing so as to induce cytotoxic T-cells, and    -   d: collecting CD⁸⁺ T cells from the co-culture of step c.

Alternatively, according to the present invention, use of the peptidesof this invention for manufacturing a pharmaceutical compositioninducing CTLs is provided. Further, the present invention also providesthe peptide of the present invention for inducing CTLs. The CD⁸⁺ T cellshaving cytotoxic activity obtained by step d can be administered to thesubject as a vaccine.

The present invention further provides isolated cytotoxic T cellsinduced using the peptides of this invention. The cytotoxic T cells,induced by stimulation with an antigen-presenting cell presenting one ormore peptides of this invention, are preferably derived from subjectswho are the target of treatment and/or prevention, and can beadministered alone or in combination with other drugs, including one ormore peptides of this invention or exosomes having anti-tumor activity.The obtained cytotoxic T cells act specifically against target cellspresenting the peptides of this invention, or preferably the samepeptide(s) used for induction. The target cells may be cells thatexpress CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10endogenously, or cells that are transfected with CDH3, EPHA4, ECT2,HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10 genes. Cells that presentthe peptides of this invention on the cell surface, due to stimulationwith these peptides, can also become targets of attack.

The present invention also provides antigen-presenting cells presentingcomplexes formed between HLA antigens and one or more peptides of thisinvention. The antigen-presenting cells, obtained through contact withthe peptides of this invention or the nucleotides encoding suchpeptides, are preferably derived from subjects who are the target oftreatment and/or prevention, and can be administered as vaccines, aloneor in combination with other drugs, including the peptides, exosomes, orcytotoxic T cells of the present invention.

The present invention also provides a composition composed of nucleicacids encoding polypeptides that are capable of forming a subunit of a Tcell receptor (TCR), and methods of using the same. The TCR subunitshave the ability to form TCRs that confer specificity to T cells fortumor cells presenting CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2,TTK or URLC10. By using the known method in the art, the nucleic acidsof alpha- and beta-chain as the TCR subunits of the CTL induced with oneor more peptides of this invention may be identified (WO2007/032255 andMorgan et al., J Immunol, 171, 3288 (2003)). The derivative TCRspreferably bind target cells displaying the CDH3, EPHA4, ECT2, HIG2,INHBB, KIF20A, KNTC2, TTK or URLC10 peptide with high avidity, andoptionally mediate efficient killing of target cells presenting theCDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK or URLC10 peptide invivo and in vitro.

The nucleic acids encoding the TCR subunits can be incorporated intosuitable vectors e.g. retroviral vectors. These vectors are well knownin the art. The nucleic acids or the vectors containing them usefullycan be transferred into a T cell, which T cell is preferably from apatient. Advantageously, the invention provides an off-the-shelfcomposition allowing rapid modification of a patient's own T cells (orthose of another mammal) to rapidly and easily produce modified T cellshaving excellent cancer cell killing properties.

Also, the present invention provides CTLs which are prepared bytransduction with the nucleic acids encoding the TCR subunitspolypeptides binding with CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2,TTK or URLC10 peptide e.g. SEQ ID NOs: 19, 22, 30, 34, 344, 358, 41, 44,46, 48, 78, 376, 379, 80, 100, 101, 110, 111, 387, 112, 394, 114, 116,117, 121, 395, 133, 135, 137, 426, 143, 147, 148, 149, 150, 152, 153,154, 156, 160, 161, 162, 163, 166, 174, 178, 186, 194, 196, 202, 210,213, 214, 217, 223, 227, 228, 233, 254, 271, 272 or 288 in the contextof HLA-A24 or HLA-A2 (HLA-A02). The transduced CTLs are capable ofhoming to cancer cells in vivo, and expanded by well known culturingmethod in vitro (e.g., Kawakami et al., J Immunol., 142, 3452-3461(1989)). The T cells of the invention can be used to form an immunogeniccomposition useful in treating or preventing cancer in a patient in needof therapy or protection (WO2006/031221).

In the context of the present invention, the term “vaccine” (alsoreferred to as an immunogenic composition) refers to a substance thatinduces anti-tumor immunity or suppresses cancers upon inoculation intoanimals. According to the present invention, polypeptides having theamino acid sequence of SEQ ID NO: 19, 22, 30, 34, 344, 358, 41, 44, 46,48, 78, 80, 100, 101, 110, 111, 387, 112, 394, 395, 133, 135, 137, 426,174, 178, 186, 194, 196, 202, 210, 213, 214, 217 or 223 were suggestedto be HLA-A24 restricted epitope peptides and those of SEQ ID NO: 376,379, 114, 116, 117, 121, 143, 147, 148, 149, 150, 152, 153, 154, 156,160, 161, 162, 163, 166, 227, 228, 233, 254, 271, 272 or 288 weresuggested to be HLA-A2 (HLA-A02) restricted epitope peptides that mayinduce potent and specific immune response against cells expressingCDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g.cancer cells expressing CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2,TTK and/or URLC10. The cancers contemplated include, but are not limitedto bladder cancer, breast cancer, cervical cancer, cholangiocellularcarcinoma, CML, colorectal cancer, endometriosis, esophageal cancer,gastric cancer, diffused type gastric cancer, liver cancer, NSCLC,lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.

Thus, the present invention also encompasses a method of inducinganti-tumor immunity using polypeptides having the amino acid sequence ofSEQ ID NO: 19, 22, 30, 34, 344, 358, 41, 44, 46, 48, 78, 376, 379, 80,100, 101, 110, 111, 387, 112, 394, 114, 116, 117, 121, 395, 133, 135,137, 426, 143, 147, 148, 149, 150, 152, 153, 154, 156, 160, 161, 162,163, 166, 174, 178, 186, 194, 196, 202, 210, 213, 214, 217, 223, 227,228, 233, 254, 271, 272 or 288 or a variant thereof (i.e., including 1,2, or several (e.g., up to 5) amino acid substitutions, deletions, oradditions). In general, anti-tumor immunity includes immune responsessuch as follows:

-   -   an induction of cytotoxic lymphocytes against tumors containing        cells expressing CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2,        TTK and/or URLC10,    -   an induction of antibodies that recognize tumors containing        cells expressing CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2,        TTK and/or URLC10, and    -   an induction of anti-tumor cytokine production.

Therefore, when a certain peptide induces any one of these immuneresponses upon inoculation into an animal, the peptide is decided tohave anti-tumor immunity inducing effect. The induction of theanti-tumor immunity by a peptide can be detected by observing in vivo orin vitro the response of the immune system in the host against thepeptide.

For example, a method for detecting the induction of cytotoxic Tlymphocytes is well known. A foreign substance that enters the livingbody is presented to T cells and B cells by the action ofantigen-presenting cells (APCs). T cells that respond to the antigenpresented by APC in antigen specific manner differentiate into cytotoxicT cells (also referred to as cytotoxic T lymphocytes or CTLs) due tostimulation by the antigen, and then proliferate; this process isreferred to herein as “activation” of T cells. Therefore, CTL inductionby a certain peptide can be evaluated by presenting the peptide to a Tcell by APC, and detecting the induction of CTL. Furthermore, APCs havethe effect of activating CD4+ T cells, CD8+ T cells, macrophages,eosinophils and NK cells. Since CD4+ T cells are also important inanti-tumor immunity, the anti-tumor immunity inducing action of thepeptide can be evaluated using the activation effect of these cells asindicators.

A method for evaluating the inducing action of CTL using dendritic cells(DCs) as APC is well known in the art. DC is a representative APC havingthe strongest CTL inducing action among APCs. In this method, the testpolypeptide is initially contacted with DC and then this DC is contactedwith T cells. Detection of T cells having cytotoxic effects against thecells of interest after the contact with DC shows that the testpolypeptide has an activity of inducing the cytotoxic T cells. Activityof CTL against tumors can be detected, for example, using the lysis of⁵¹Cr-labeled tumor cells as the indicator. Alternatively, it is wellknown to evaluate the degree of tumor cell damage using 3H-thymidineuptake activity or LDH (lactose dehydrogenase)-release as the indicator.Furthermore, it can be also examined by measuring IFN-gamma produced andreleased by CTL in the presence of antigen-presenting cells that carryimmobilized peptides by visualizing using anti-IFN-gamma antibodies,such as an ELISPOT assay.

Apart from DC, peripheral blood mononuclear cells (PBMCs) may also beused as the APC. The induction of CTL is reported to be enhanced byculturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL hasbeen shown to be induced by culturing PBMC in the presence of keyholelimpet hemocyanin (KLH) and IL-7.

The test polypeptides confirmed to possess CTL inducing activity bythese methods are polypeptides having DC activation effect andsubsequent CTL inducing activity. Therefore, polypeptides that induceCTL against cells expressed CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A,KNTC2, TTK and/or URLC10 are useful as vaccines against diseasesassociating CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/orURLC10, e.g. cancers. Furthermore, APC that have acquired the ability toinduce CTL against a disease associated with the over-expression ofCDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g.cancers, by contacting with the polypeptides are useful as vaccinesagainst the disease. Furthermore, CTL that have acquired cytotoxicitydue to presentation of the polypeptide antigens by APC can be also usedas vaccines against a disease associating CDH3, EPHA4, ECT2, HIG2,INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers. Such therapeuticmethods for a disease associating CDH3, EPHA4, ECT2, HIG2, INHBB,KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers, using anti-tumorimmunity due to APC and CTL, are referred to as cellular immunotherapy.The cancers contemplated include, but are not limited to, bladdercancer, breast cancer, cervical cancer, cholangiocellular carcinoma,CML, colorectal cancer, endometriosis, esophageal cancer, gastriccancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renalcarcinoma, SCLC, soft tissue tumor and testicular tumor.

Generally, when using a polypeptide for cellular immunotherapy,efficiency of the CTL-induction can be increased by combining aplurality of polypeptides having different structures and contactingthem with DC. Therefore, when stimulating DC with protein fragments, itis advantageous to use a mixture of multiple types of fragments.

The induction of anti-tumor immunity by a polypeptide can be furtherconfirmed by observing the induction of antibody production againsttumors. For example, when antibodies against a polypeptide are inducedin a laboratory animal immunized with the polypeptide, and when growth,proliferation and/or metastasis of tumor cells is suppressed by thoseantibodies, the polypeptide is determined to induce anti-tumor immunity.

Anti-tumor immunity can be induced by administering a vaccine of thisinvention, and the induction of anti-tumor immunity enables treatmentand prevention of a disease associated with the over-expression of CDH3,EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g.cancers. Therapy against or prevention of the onset of a diseaseassociated with the over-expression of CDH3, EPHA4, ECT2, HIG2, INHBB,KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers, may include inhibitionof the growth of cells expressing CDH3, EPHA4, ECT2, HIG2, INHBB,KIF20A, KNTC2, TTK and/or URLC10, e.g. cancer cells, involution of thesecells and suppression of occurrence of these cells, e.g. cancer cells.Decrease in mortality of individuals having a disease associating CDH3,EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g.cancers, decrease of the disease markers in the blood, alleviation ofdetectable symptoms accompanying the disease and such are also includedin the therapy or prevention of the disease, e.g. cancers. Suchtherapeutic and preventive effects are preferably statisticallysignificant, for example, observed at a significance level of 5% orless, wherein the therapeutic or preventive effect of a vaccine againsta disease associating CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTKand/or URLC10, e.g. cancers, is compared to a control without vaccineadministration. For example, Student's t-test, the Mann-Whitney U-testor ANOVA may be used for determining statistical significance.

In that the present invention provides a method for treating, orpreventing a disease associated with the over-expression of CDH3, EPHA4,ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers, thetherapeutic compounds or compositions may be administeredprophylactically or therapeutically to subjects suffering from or atrisk of (or susceptible to) developing the disease. Such subjects may beidentified using standard clinical methods. In the context of thepresent invention, prophylactic administration occurs prior to themanifestation of overt clinical symptoms of disease, such that a diseaseor disorder is prevented or alternatively delayed in its progression. Inthe context of the field of medicine, the term “prevent” encompasses anyactivity which reduces the burden of mortality or morbidity fromdisease. Prevention can occur at primary, secondary and tertiaryprevention levels. While primary prevention avoids the development of adisease, secondary and tertiary levels of prevention encompassactivities aimed at preventing the progression of a disease and theemergence of symptoms as well as reducing the negative impact of analready established disease by restoring function and reducingdisease-related complications.

In the context of cancer treatment, the term “efficacious” refers to atreatment that leads to a decrease in size, prevalence or metastaticpotential of cancer in a subject. When a treatment is appliedprophylactically, “efficacious” means that the treatment retards orprevents occurrence of cancer or alleviates a clinical symptom ofcancer. The assessment of cancer can be made using standard clinicalprotocols. Furthermore, the efficaciousness of a treatment may bedetermined in association with any known method for diagnosing ortreating cancer. For example, cancer can be diagnosedhistopathologically or by identifying symptomatic anomalies.

The above-mentioned peptide, having immunological activity, or apolynucleotide or vector encoding such a peptide, may be combined withan adjuvant. An adjuvant refers to a compound that enhances the immuneresponse against the peptide when administered together (orsuccessively) with the peptide having immunological activity. Examplesof suitable adjuvants include cholera toxin, salmonella toxin, alum andsuch, but are not limited thereto. Furthermore, a vaccine of thisinvention may be combined appropriately with a pharmaceuticallyacceptable carrier. Examples of such carriers are sterilized water,physiological saline, phosphate buffer, culture fluid and such.Furthermore, the vaccine may contain as necessary, stabilizers,suspensions, preservatives, surfactants and such. The vaccine isadministered systemically or locally. Vaccine administration may beperformed by single administration or boosted by multipleadministrations.

When using APC or CTL as the vaccine of this invention, a diseaseassociated with the over-expression of CDH3, EPHA4, ECT2, HIG2, INHBB,KIF20A, KNTC2, TTK and/or URLC10, e.g. cancers, can be treated orprevented, for example, by the ex vivo method. More specifically, PBMCsof the subject receiving treatment or prevention are collected,contacted ex vivo with a peptide of the present invention. Following theinduction of APC or CTL, the cells may be administered to the subject.APC can be also induced by introducing a vector encoding the peptideinto PBMCs ex vivo. APC or CTL induced in vitro can be cloned prior toadministration. By cloning and growing cells having high activity ofdamaging target cells, cellular immunotherapy can be performed moreeffectively. Furthermore, APC and CTL isolated in this manner may beused for cellular immunotherapy not only against individuals from whomthe cells are derived, but also against similar types of diseases inother individuals.

Aspects of the present invention are described in the followingexamples, which are presented only to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below.

EXAMPLES

Hereinafter, the present invention is exemplified, but not restricted,by the following Examples. However, materials, methods and suchdescribed herein only illustrate aspects of the invention and in no wayare intended to limit the scope of the present invention. As such,materials, methods and such similar or equivalent to those describedtherein may be used in the practice or testing of the present invention.

Example 1 Materials and Methods Cell Lines

A24-LCL cells (HLA-A24), human B-lymphoblastoid cell line, wasestablished by transforming with Epstain-barr virus. T2 cell, COS7,A498, Caki-2 and HEK 293 were purchased from ATCC. Caki-1 and MIAPaca-2were purchased from JCRB. PK-45P, PK-59, TE-5 and TE-6 were purchasedfrom TKG. 293 T was purchased from GenHunter.

Candidate Selection of Peptide Derived from CDH3, EPHA4, ECT2, HIG2,INHBB, KIF20A, KNTC2, TTK and URLC10

9-mer and 10-mer peptides derived from CDH3, EPHA4, ECT2, HIG2, INHBB,KIF20A, KNTC2, TTK or URLC10 that bind to HLA-A*2402 or HLA-A*0201molecule were predicted using the binding prediction software “BIMAS”(http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform) (ParkerK C, et al., (1994) J Immunol.; 152(1):163-75.; Kuzushima K, et al.,(2001) Blood.; 98(6):1872-81.). These peptides were synthesized by Sigma(Sapporo, Japan) according to the standard solid phase synthesis methodand purified by reversed phase HPLC. The purity (>90%) and the identityof the peptides were determined by analytical HPLC and mass spectrometryanalysis, respectively. Peptides were dissolved in dimethylsulfoxide(DMSO) at 20 mg/ml and stored at −80 degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as antigen-presentingcells (APCs) to induce CTL responses against peptides presented on HLA.DCs were generated in vitro as described elsewhere (Nukaya I et al.,(1999) Int. J. Cancer 80, 92-7., Tsai V et al., (1997) J. Immunol158:1796-802.). Briefly, peripheral blood mononuclear cells (PBMCs)isolated from a normal volunteer (HLA-A*2402 and/or HLA-A*0201) byFicoll-Paque (Pharmacia) solution were separated by adherence to aplastic tissue culture flask (Becton Dickinson) so as to enrich them forthe monocyte fraction. The monocyte-enriched population was cultured inthe presence of 1000 U/ml of GM-CSF (Genzyme) and 1000 U/ml of IL-4(Genzyme) in AIM-V(Invitrogen) containing 2% heat-inactivated autologousserum (AS). After 7 days in the culture, the cytokine-generated DCs werepulsed with 20 micro g/ml of the synthesized peptides in the presence of3 micro g/ml of beta 2-microglobulin for 4 hrs at 20 degrees C. inAIM-V. These peptide-pulsed DCs were then inactivated by MMC (30 microg/ml for 30 mins) and mixed at a 1:20 ratio with autologous CD8⁺ Tcells, obtained by positive selection with Dynabeads M-450 CD8 (Dynal)and DETACHa BEAD™ (Dynal). These cultures were set up in 48-well plates(Corning); each well contained 1.5×10⁴ peptide-pulsed DCs, 3×10⁵ CD8⁺ Tcells and 10 ng/ml of IL-7 (Genzyme) in 0.5 ml of AIM-V/2% AS. Threedays later, these cultures were supplemented with IL-2 (CHIRON) to afinal concentration of 20 IU/ml. On day 7 and 14, the T cells werefurther restimulated with the autologous peptide-pulsed DCs. The DCswere prepared each time by the same way described above. CTL was testedagainst peptide-pulsed A24-LCL cells or T2 cells after the 3rd round ofpeptide stimulation on day 21.

CTL Expansion Procedure

CTLs were expanded in culture using the method similar to that describedby Riddell S R, et al., (Walter E A et al., (1995) N Engl J Med333:1038-44.; Riddel S R, et al., (1996) Nature Med. 2:216-23.). A total5×10⁴ of CTLs were resuspended in 25 ml of AIM-V/5% AS with 2 kinds ofhuman B-lymphoblastoid cell lines, inactivated by MMC, in the presenceof 40 ng/ml of anti-CD3 monoclonal antibody (Pharmingen). One day afterinitiating the cultures, 120 IU/ml of IL-2 were added to the cultures.The cultures were fed with fresh AIM-V/5% AS containing 30 IU/ml of IL-2on days 5, 8 and 11.

Establishment of CTL Clones

The dilutions were made to have 0.3, 1, and 3 CTLs/well in 96round-bottomed micro titer plate (Nalge Nunc International). CTLs werecultured with 7×10⁴ cells/well of 2 kinds of human B-lymphoblastoid celllines, 30 ng/ml of anti-CD3 antibody, and 125 U/ml of IL-2 in total of150 micro 1/well of AIM-V containing 5% AS. 50 micro 1 /well of IL-2 wasadded to the medium 10 days later so that IL-2 became 125 U/ml in thefinal concentration. CTL activity of CTLs was tested on the 14th day,and CTL clones were expanded using the same method above.

Specific CTL Activity

To examine the specific CTL activity, IFN-gamma ELISPOT assay andIFN-gamma ELISA assay were performed. Briefly, peptide-pulsed A24-LCL orT2 cell (1×10⁴/well) was prepared as stimulator cells. Cultured Cells in48 wells or CTL clones after limiting dilution were used as respondercells. IFN-gamma ELISPOT assay and ELISA assay were performed undermanufacture procedure.

Establishment of the Cells Forcibly Expressing Either or Both of theTarget Gene and HLA-A02 or HLA-A24

The cDNA encoding an open reading frame of taget genes or HLA-A02 orHLA-A24 was amplified by PCR. The PCR-amplified product was cloned intopcDNA3.1 myc-His vector (Invitrogen). The plasmids were transfected intothe taget cells, HLA-A02 and HLA-A24-null normal human cell line COS7 or293T using lipofectamine (Invitrogen) according to the manufacturer'srecommended procedures. Alternatively, the plasmid contained the targetgenes were transfected into A24-LCL by electroporation usingGenePulserII (Biorad). Briefly, 2.5×10⁶ A24-LCL cells were pulsed with10 mcg prasmid at 140V and 1000 micro F. After 2 days from transfection,the transfected cells were treated with Cell dissociation solution andused as the target cells for CTL activity assay.

Cytotoxicity Assay

Cytotoxic activity was evaluated by a four-hour ⁵¹Cr release assay. Thetarget cells were pulsed with a 20 micro g/mL concentration of peptideovernight. The target cells were labeled with 100 micro Ci of Na₂ ⁵¹CrO₄at 37 degrees C. for one hour, and then washed three times withRPMI1640. The target cells (1×10⁴/100 micro L) and 100 micro L ofeffector cells at various numbers with a total volume of 200 micro Lwere placed into a round-bottomed 96-well microtiter plate (Corning),and cultured at 37 degrees C. in a CO₂ incubator for four hours. Afterculturing, 100 micro L of the supernatant was collected from each well,and measured the radioactivity using a gamma counter. Spontaneousrelease was the radioactivity from the target cells with medium in theabsence of effector cells, and maximum release was the radioactivityfrom the target cells with 1 M HCl.

The Percentage of specific cytotoxicity was determined by calculating asfollowing formula:

% Specific lysis=[(experimental release−spontaneous release)/(maximumrelease−spontaneous release)]×100.

Results Enhanced CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK andURLC10 Expression in Cancers

The global gene expression profile data obtained from various cancersusing cDNA-microarray revealed that the expression of the followinggenes was elevated.

-   -   CDH3 (GenBank Accession No. NM_001793; SEQ ID Nos.1, 2),    -   EPHA4 (GenBank Accession No. L36645; SEQ ID Nos.3, 4),    -   ECT2 (GenBank Accession No. AY376439; SEQ ID Nos.5, 6),    -   HIG2 (GenBank Accession No. NM_013332; SEQ ID Nos.7, 8),    -   INHBB (GenBank Accession No. NM_002193; SEQ ID Nos.9, 435, 10,        436),    -   KIF20A (GenBank Accession No. NM_005733; SEQ ID Nos.11, 12),    -   KNTC2 (GenBank Accession No. AF017790; SEQ ID Nos.13, 14),    -   TTK (GenBank Accession No. NM_003318; SEQ ID Nos.15, 16) and    -   URLC10 (GenBank Accession No. NM_017527; SEQ ID Nos.17, 18)    -   CDH3 expression was validly elevated in the following cancers in        comparison with corresponding normal tissue.    -   26 out of 34 bladder cancer,    -   17 out of 19 cervical cancer,    -   19 out of 19 cholangiocellular carcinoma,    -   30 out of 34 colorectal cancer,    -   20 out of 21 endometriosis,    -   13 out of 20 gastric cancer,    -   7 out of 8 diffuse-type gastric cancer,    -   36 out of 37 NSCLC,    -   16 out of 16 pancreatic cancer,    -   21 out of 21 soft tissue tumor and    -   10 out of 10 testicular tumor

EPHA4 expression was validly elevated in the following cancers incomparison with corresponding normal tissue.

-   -   14 out of 34 bladder cancer,    -   8 out of 14 cervical cancer,    -   10 out of 25 cholangiocellular carcinoma,    -   5 out of 15 endometriosis,    -   5 out of 8 diffuse-type gastric cancer,    -   5 out of 5 ovarian cancer,    -   14 out of 14 pancreatic cancer, 20 out of 51 prostate cancer and        14 out of 23 soft tissue tumor

ECT2 expression was validly elevated in the following cancers incomparing with corresponding normal tissue.

-   -   17 out of 19 bladder cancer,    -   5 out of 12 breast cancer,    -   14 out of 14 cervical cancer,    -   13 out of 13 cholangiocellular carcinoma,    -   5 out of 5 CML,    -   7 out of 8 colorectal cancer,    -   12 out of 16 esophageal cancer,    -   6 out of 16 NSCLC,    -   8 out of 10 lymphoma,    -   1 out of 1 pancreatic cancer,    -   10 out of 13 prostate cancer,    -   3 out of 6 renal carcinoma and    -   12 out of 13 SCLC cancer    -   HIG2 expression was validly elevated in 19 out of 20 renal        cancer and 7 out of 9 soft tissue tumor in comparing with        corresponding normal tissue.    -   INHBB expression was validly elevated in the following cancers        in comparing with corresponding normal tissue.    -   10 out of 21 cholangiocellular carcinoma,    -   12 out of 12 esophageal cancer,    -   10 out of 13 NSCLC,    -   22 out of 24 renal carcinoma,    -   8 out of 14 SCLC cancer and    -   45 out of 49 soft tissue tumor

KIF20A expression was validly elevated in the following cancers incomparing with corresponding normal tissue.

-   -   31 out of 31 bladder cancer,    -   38 out of 61 breast cancer,    -   10 out of 11 cholangiocellular carcinoma,    -   7 out of 19 esophageal cancer,    -   21 out of 22 NSCLC,    -   6 out of 6 ovarian cancer,    -   17 out of 36 prostate cancer,    -   6 out of 11 renal carcinoma and    -   15 out of 15 SCLC

KNTC2 expression was validly elevated in the following cancers incomparing with corresponding normal tissue.

-   -   30 out of 32 bladder cancer,    -   47 out of 56 breast cancer,    -   10 out of 10 cervical cancer,    -   16 out of 22 cholangiocellular carcinoma,    -   17 out of 37 CML,    -   3 out of 10 colorectal cancer,    -   11 out of 46 esophagus cancer,    -   15 out of 19 NSCLC,    -   7 out of 8 lymphoma,    -   20 out of 24 osteosarcoma,    -   3 out of 5 ovarian cancer,    -   2 out of 2 pancreatic cancer,    -   15 out of 37 prostate cancer,    -   14 out of 19 renal carcinoma,    -   15 out of 15 SCLC and    -   40 out of 59 soft tissue tumor

TTK expression was validly elevated in the following cancers incomparing with corresponding normal tissue.

-   -   27 out of 27 bladder cancer,    -   25 out of 30 breast cancer,    -   15 out of 16 cervical cancer,    -   10 out of 10 cholangiocellular carcinoma,    -   5 out of 7 CML,    -   6 out of 10 colorectal cancer,    -   24 out of 44 esophageal cancer,    -   8 out of 15 liver cancer,    -   12 out of 12 NSCLC,    -   6 out of 6 lymphoma,    -   13 out of 16 osteoblastoma,    -   12 out of 17 prostate cancer,    -   15 out of 15 SCLC and    -   16 out of 33 soft tissue tumor

URLC10 expression was validly elevated in the following cancers incomparing with corresponding normal tissue

-   -   29 out of 29 bladder cancer,    -   15 out of 16 cervical cancer,    -   7 out of 7 cholangiocellular carcinoma,    -   7 out of 19 esophageal cancer,    -   3 out of 3 gastric cancer, 24 out of 27 NSCLC,    -   15 out of 19 osteosarcoma,    -   4 out of 5 pancreatic cancer,    -   33 out of 43 soft tissue tumor.

TABLE 1 Ratio of cases observed up-regulation of CDH3, EPHA4, ECT2,HIG2, INHBB, KIF20A, KNTC2, TTK or URLC10 in cancerous tissue ascompared to normal corresponding tissue CDH3 EPHA4 ECT2 HIG2 INHBBBladder cancer 26/34 14/34 17/19 — — Breast cancer — —  5/12 — —Cervical cancer 17/19  8/14 14/14 — — Cholangiocellularcarcinoma 19/1910/25 13/13 — 10/21 CML — — 5/5 — — Colectal cancer 30/34 — 7/8 — —Endometriosis 20/21  5/15 — — — Esophageal cancer — — 12/16 — 12/12Gastric camcer 13/20 — — — — Diffuse-type Gastric cancer 7/8 5/8 — — —Liver cancer — — — — — non-small cell lung cancer 36/37 —  6/16 — 10/13Lymphoma — —  8/10 — — Osteosarcoma — — — — — Ovarian cancer — 5/5 — — —Pancreatic cancer 16/16 14/14 1/1 — — Prostate cancer — 20/51 10/13 — —Renal carcinoma — — 3/6 19/20 22/24 Small cell lung cancer — — 12/13 — 8/14 Soft tissue tumor 21/21 14/23 — 7/9 45/49 Testicular tumor 10/10 —— — — KIF20A KNTC2 TTK URLC10 Bladder cancer 31/31 30/32 27/27 29/29Breast cancer 38/61 47/56 25/30 — Cervical cancer — 10/10 15/16 15/16Cholangiocellularcarcinoma 10/11 16/22 10/10 7/7 CML — 17/37 5/7 —Colectal cancer —  3/10  6/10 — Endometriosis — — — — Esophageal cancer 7/19 11/46 24/44  7/19 Gastric camcer — — — 3/3 Diffuse-type Gastriccancer — — — — Liver cancer — —  8/15 — non-small cell lung cancer 21/2215/19 12/12 24/27 Lymphoma — 7/8 6/6 — Osteosarcoma — 20/24 13/16 15/19Ovarian cancer — 3/5 — — Pancreatic cancer 6/6 2/2 — 4/5 Prostate cancer17/36 15/37 12/17 — Renal carcinoma  6/11 14/19 — — Small cell lungcancer 15/15 15/15 15/15 — Soft tissue tumor — 40/59 16/33 33/43Testicular tumor — — — —Prediction of HLA-A24 or HLA-A2 (HLA-A02) Binding Peptides Derived fromCDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK or URLC10

Table 2 sets forth the HLA-A*2402 binding peptides for CDH3 in order ofbinding affinity. Table 2A sets forth 9-mer peptides derived from CDH3and Table 2B sets forth 10-mer peptides derived from CDH3.

Table 3 sets forth the HLA-A*2402 and HLA-A*0201 binding peptides forEPHA4 in order of binding affinity. Table 3A sets forth the HLA-A*2402binding 9-mer peptides derived from EPHA4, Table 3B shows the HLA-A*2402binding 10-mer peptides derived from EPHA4 and Table 3C sets forth theHLA-A*0201 binding 9-mer peptides derived from EPHA4.

Table 4 sets forth the HLA-A*2402 binding peptides for ECT2 in order ofbinding affinity. Table 4A sets forth 9-mer peptides derived from ECT2and Table 4B shows 10-mer peptides derived from ECT2.

Table 5 sets forth the HLA-A*2402 and HLA-A*0201 binding peptides forHIG2, Table 5A sets forth the HLA-A*2402 binding 9-mer peptides derivedfrom HIG2, Table 5B sets forth the HLA-A*2402 binding 10-mer peptidesderived from HIG2, Table 5C sets forth the HLA-A*0201 binding 9-merpeptides derived from HIG2, and Table 5D sets forth HLA-A*0201 binding10-mer peptides derived from HIG2.

Table 6 sets forth the HLA-A*2402 and HLA-A*0201 binding peptides forINHBB, Table 6A shows the HLA-A*2402 binding 9-mer peptides derived fromINHBB, Table 6B sets forth the HLA-A*2402 binding 10-mer peptidesderived from INHBB, Table 6C sets forth the HLA-A*0201 binding 9-merpeptides derived from INHBB, and Table 6D sets forth HLA-A*0201 binding10-mer peptides derived from INHBB.

Table 7 sets forth the HLA-A*2402 binding peptides for KIF20A in orderof binding affinity. Table 7A sets forth 9-mer peptides derived fromKIF20A and Table 7B sets forth 10-mer peptides derived from KIF20A.

Table 8 sets forth the HLA-A*2402 binding peptides for KNTC2 in order ofbinding affinity. Table 8A sets forth 9-mer peptides derived from KNTC2and Table 8B sets forth 10-mer peptides derived from KNTC2.

Table 9 sets forth the HLA-A*0201 binding peptides for TTK in order ofbinding affinity. Table 9A sets forth 9-mer peptides derived from TTKand Table 9B sets forth 10-mer peptides derived from TTK.

Table 10 sets forth the HLA-A*0201 binding peptides for URLC10 in orderof binding affinity. Table 10A sets forth 9-mer peptides derived fromURLC10 and Table 10B sets forth 10-mer peptides derived from URLC10.

Explanation and definition about the terms in tables

Start position indicates the number of amino acid from N-terminal.Binding score is derived from “BIMAS” described in Materials andMethods.Positive donor number indicates the number of donors whose CD8+-T-cellscan be induced to the specific CTL by the ex vivo stimulation withantigen-presenting cells. This is shown as (positive donor number/wholedonor number).Positive well number indicates the number of wells where specificIFN-gamma production can be detected by IFN-gamma ELISPOT assay. 4 to 8wells can be prepared from one donor. This is shown as (positive wellsnumber/the number of whole wells tested by IFN-gamma ELISPOT assay).Positive CTL line indicates the number of CTL line established frompositive wells. The generation of CTL line is determined by ELISA. Thisis shown as (established CTL line number/the number of positive wellstested by IFN-gamma ELISPOT assay).No positive donor is not defined by no detectable positive wells, but byno established CTL line.The peptides showed by bold character in tables possesses thestimulation activity of the T cells. No data at positive donor number,positive well number and positive CTL line indicating “−” means that thepeptides can't be synthesized for any reason.

TABLE 2A HLA-A*2402 binding 9-mer peptides derived from CDH3 PositivePositive Strat Amino acid Binding donor well Positive SEQ ID positionsequence Score number number CTL line NO. 513 IYEVMVLAM 37.5 1/3 19 667LFLLLVLLL 36 — — — 20 30 VFREAEVTL 24 0/3  1/22 0/1 21 406 LYVEVTNEA16.632 1/3 22 332 KYEAHVPEN 16.5 0/3  1/22 0/1 23 180 KYELFGHAV 15 0/3 1/22 0/1 24 85 RSLKERNPL 14.4 0/3  1/22 0/1 25 5 RGPLASLLL 12 0/3  2/220/2 26 652 KGGFILPVL 11.2 0/3  0/22 — 27 248 TYNGVVAYS 10.5 0/3  2/220/2 28 65 LFSTDNDDF 10 0/3  0/22 — 29 94 KIFPSKRIL 9.6 0/1 0/8 — 306 221RGSVLEGVL 9.6 0/1 0/8 — 307 668 FLLLVLLLL 8.4 — — — 308 754 IGNFIIENL8.4 — — — 309 311 TAVAVVEIL 8.4 0/1 0/8 — 310 557 NQSPVRQVL 8.064 0/10/8 — 311 611 KQDTYDVHL 8 0/1 0/8 — 312 781 DYEGSGSDA 7.5 0/1 0/8 — 313165 GWLLLNKPL 7.2 0/1 0/8 — 314 656 ILPVLGAVL 7.2 0/1 0/8 — 315 770TAPPYDTLL 7.2 0/1 0/8 — 316 602 VVLSLKKFL 7.2 0/1 0/8 — 317 665ALLFLLLVL 7.2 — — — 318 410 VTNEAPFVL 7.2 0/1 0/8 — 319 662 AVLALLFLL7.2 — — — 320 613 DTYDVHLSL 6.72 0/1 0/8 — 321 6 GPLASLLLL 6 0/1 0/8 —322 564 VLNITDKDL 6 0/1 0/8 — 323 159 AVEKETGWL 6 0/1 0/8 — 324 511NNIYEVMVL 6 0/1 0/8 — 325 11 LLLLQVCWL 6 — — — 326 57 GCPGQEPAL 6 0/10/8 — 327 293 EYTLTIQAT 6 0/1 0/8 — 328 79 ETVQERRSL 6 0/1 0/8 — 329 475SYRILRDPA 6 0/1 0/8 — 330 493 GQVTAVGTL 6 0/1 0/8 — 331 661 GAVLALLFL 60/1 0/8 — 332 388 GILTTRKGL 6 0/1 0/8 — 333 382 HPESNQGIL 6 0/1 0/8 —334 663 VLALLFLLL 5.76 — — — 335 598 EGDTVVLSL 5.6 0/1 0/8 — 336 278TISVISSGL 5.6 0/1 2/8 0/2 337 659 VLGAVLALL 5.6 0/1 0/8 — 338 811EWGSRFKKL 5.28 0/1 0/8 — 339 445 KVVEVQEGI 5.04 0/1 0/8 — 340 614TYDVHLSLS 5 0/1 0/8 — 341 142 FYSITGPGA 5 0/1 0/8 — 342 246 IYTYNGVVA 50/1 0/8 — 343

TABLE 2B HLA-A*2402 binding 10-mer peptides derived from CDH3 positivepositive strat Binding donor well positive SEQ ID position sequenceScore number number CTL line NO 807 DYLNeWGSRF 150 1/3 30 248 TYNGvVAYSI105 0/3 4/22 0/4 31 667 LFLLlVLLLL 42 — — — 32 397 DFEAkNQHTL 30 0/32/22 0/2 33 332 KYEAhVPENA 21 1/3 34 180 KYELFGHAVS 15 0/3 2/22 0/2 35510 RNNIYEVMVL 12 0/3 4/22 0/4 36 5 RGPLASLLLL 12 0/3 1/22 0/1 37 477RILRDPAGWL 12 0/3 1/22 0/1 38 556 CNQSPVRQVL 10.08 0/3 2/22 0/2 39 655FILPvLGAVL 8.64 1/3 344 662 AVLAlLFLLL 8.64 — — — 345 277 GTISvISSGL 8.40/3 0/20 — 346 781 DYEGsGSDAA 7.5 0/3 0/20 — 347 601 TVVLsLKKFL 7.2 0/33/20 0/3 348 158 FAVEkETGWL 7.2 0/3 0/20 — 349 665 ALLFILLVLL 7.2 — — —350 259 SQEPkDPHDL 7.2 0/3 0/20 — 351 664 LALLfLLLVL 7.2 — — — 352 42GAEQePGQAL 7.2 0/3 1/20 0/1 353 661 GAVLaLLFLL 7.2 — — — 354 595VNEEgDTVVL 7.2 0/2 0/12 — 355 340 NAVGhEVQRL 7.2 0/2 0/12 — 356 411TNEApFVLKL 6.6 0/2 0/12 — 357 470 ENQKiSYRIL 6 1/2 358 10 SLLLlQVCWL 60/2 1/12 0/1 359 721 GLEArPEVVL 6 0/2 2/12 0/2 360 345 EVQRlTVTDL 6 0/24/12 0/4 361 2 GLPRgPLASL 6 0/2 3/12 0/3 362 657 LPVLgAVLAL 6 — — — 363563 QVLNiTDKDL 6 0/2 1/12 0/1 364 159 AVEKeTGWLL 6 0/2 2/12 0/2 365 492SGQVtAVGTL 6 0/2 — — 366 387 QGILtTRKGL 6 0/2 — — 367 525 SPPTtGTGTL 60/2 2/12 0/2 368 358 NSPAwRATYL 6 0/2 2/12 0/2 369 122 GPFPqRLNQL 5.760/2 3/12 0/3 370 753 EIGNfIIENL 5.6 0/2 1/12 0/1 371 310 TTAVaVVEIL 5.6— — — 372 246 IYTYnGVVAY 5 0/2 2/12 0/2 373 805 DYDYlNEWGS 5 0/2 0/12 —374

TABLE 3A HLA-A*2402 binding 9-mer peptides derived from EPHA4 positivepositive strat Binding donor well positive SEQ ID position sequenceScore number number CTL line NO 97 VYIEIKFTL 504 0/2 1/16 0/1 40 453RYSVALAWL 400 2/3 41 25 VYPANEVTL 300 0/3 0/22 — 42 384 HYTPQQNGL 2880/3 1/22 0/1 43 5 FYFALFSCL 288 1/2 44 519 GYGDFSEPL 240 0/3 3/22 0/3 45869 KFGQIVNML 67.2 1/3 46 777 AYTTRGGKI 55 0/3 1/22 0/1 47 420 KYNPNPDQS18 1/3 48 749 RNILVNSNL 16.8 0/3 1/22 0/1 49 734 KYLSDMSYV 15 0/3 0/22 —50 879 KLIRNPNSL 14.4 0/3 0/22 — 51 926 RYKDNFTAA 14.4 0/3 0/22 — 52 834KAIEEGYRL 14.4 0/3 0/22 — 53 574 KYSKAKQEA 13.2 0/3 0/22 — 54 184AFQDVGACI 12.6 0/3 1/22 0/1 55 252 WLVPIGNCL 12.096 0/3 0/22 — 56 326RPPSAPLNL 12 0/3 0/22 — 57 203 KCPLTVRNL 12 0/3 0/22 — 58 360 SYNVVCKKC11.55 0/3 0/22 — 59

TABLE 3B HLA-A*2402 binding 10-mer peptides derived from EPHA4 positivepositive strat Binding donor well positive SEQ ID position sequenceScore number number CTL line NO 25 VYPANEVTLL 300 0/3 0/22 — 60 244MYCGADGEWL 200 0/3 1/22 0/1 61 657 GYTDKQRRDF 120 0/3 1/22 0/1 62 5FYFAlFSCLF 100 — — — 63 102 KFTLRDCNSL 48 0/3 1/22 0/1 64 818 SYGERPYWDM30 0/3 2/22 0/2 65 4 IFYFALFSCL 28.8 — — — 66 808 SYGIVMWEVM 25 — — — 67630 EFGEVCSGRL 24 0/3 0/22 — 68 420 KYNPNPDQSV 21.6 0/3 0/22 — 69 930NFTAAGYTTL 20 0/2 0/16 — 70 675 QFDHPNIIHL 20 0/3 0/22 — 71 708AFLRKNDGRF 15 0/3 0/22 — 72 579 KQEADEEKHL 12 0/3 1/22 0/1 73 727RGIGSGMKYL 12 0/3 0/22 — 74 96 RVYIEIKFTL 11.2 0/2 1/16 0/1 75 507SYVFHVRART 10.5 0/3 1/22 0/1 76 251 EWLVPIGNCL 10.08 0/3 0/22 — 77 24RVYPANEVTL 9.6 1/3 78 699 EYMENGSLDA 9 0/3 0/22 — 79

TABLE 3C HLA-A*0201 binding 9-mer peptides derived from EPHA4 positivepositive strat Binding donor well positive SEQ ID position sequenceScore number number CTL line NO 8 ALFSCLFGI 514.942 — — — 375 501GLNPLTSYV 382.536 1/1 376 12 CLFGICDAV 126.098 0/1 1/5 0/1 377 977QMHGRMVPV 115.534 0/1 1/5 0/1 378 165 KLNTEIRDV 111.979 1/1 379 252WLVPIGNCL 98.267 0/1 1/5 0/1 380 879 KLIRNPNSL 74.768 0/1 1/5 0/1 381559 VVILIAAFV 56.902 — — — 382 812 VMWEVMSYG 39.386 0/1 0/5 — 383 728GIGSGMKYL 37.157 0/1 0/5 — 384 750 NILVNSNLV 35.385 0/1 1/5 0/1 385 937TTLEAVVHV 33.705 0/1 1/5 0/1 386

TABLE 4A HLA-A*2402 binding 9-mer peptides derived from ECT2 positivepositive strat Binding donor well positive SEQ ID position sequenceScore number number CTL line NO 515 TYPPFVNFF 216 1/1 80 140 LYCTSMMNL200 0/1 0/8 — 81 298 LYVVKQEWF 150 0/1 0/8 — 82 435 NYVNILATI 105 0/10/8 — 83 773 IYTADPESF 100 0/1 0/8 — 84 110 LYKADCRVI 50 0/1 0/8 — 85739 SFQMTSDEL 33 0/1 0/8 — 86 504 IFLKYSKDL 30 0/1 0/8 — 87 867FFERRSHTL 30 0/1 0/8 — 88 178 DFNSKVTHL 30 0/1 0/8 — 89 61 KQEELIKAL17.28 0/1 0/8 — 90 657 RGEQVTLFL 16.8 0/1 2/8 0/2 91 568 RLPSVALLL 16.80/1 0/8 — 92 550 KPECGRQSL 14.4 0/1 0/8 — 93 470 IFGSIPDIF 14 0/1 0/8 —94 116 RVIGPPVVL 12 0/1 0/8 — 95 507 KYSKDLVKT 11 0/1 0/8 — 96 223DFYAAVDDF 10 0/1 0/8 — 97

TABLE 4B HLA-A*2402 binding 10-mer peptides derived from ECT2 positivepositive strat Binding donor well positive SEQ ID position sequenceScore number number CTL line NO 322 LYEKaNTPEL 330 0/1 0/8 — 98 435NYVNiLATII 90 0/1 0/8 — 99 40 SYVEeEMPQI 90 1/1 100 101 DFQDsVFNDL72.576 1/1 101 866 SFFErRSHTL 24 0/1 0/8 — 102 811 SFSKtPKRAL 20 0/1 1/80/1 103 268 KYLPlGDERC 18 0/1 0/8 — 104 84 EFEGlDSPEF 16.5 0/1 1/8 0/1105 236 KVPPfQDCIL 14.4 0/1 0/8 — 106 728 RPPTeQANVL 14.4 0/1 0/8 — 107507 KYSKdLVKTY 12 0/1 0/8 — 108 281 VVEEnIVKDL 10.08 0/1 0/8 — 109

TABLE 5A HLA-A*2402 binding 9-mer peptides derived from HIG2 positivepositive strat Binding donor well positive SEQ ID position sequenceScore number number CTL line NO 19 IFVRVMESL 42 1/3 110 22 RVMESLEGL14.4 1/3 111 8 YLLGVVLTL 8.4 1/3 387 7 LYLLGVVLT 7.5 0/2 3/15 0/3 388 23VMESLEGLL 7.2 0/2 0/16 — 389 9 LLGVVLTLL 5.6 — — — 390

TABLE 5B Table 5B HLA-A*2402 binding 10-mer peptides derived from HIG2posi- posi- strat tive tive SEQ posi- Binding donor well positive IDtion sequence Score number number CTL line NO 7 LYLLGVVLTL 420 1/3 11222 RVMESLEGLL 17.28 0/3 4/24 0/4 113 8 YLLGVVLTLL 8.4 — — — 391 5LNLYLLGVVL 7.2 0/2 0/12 — 392 46 LANTEPTKGL 6 0/2 0/14 — 393 18SIFVRVMESL 5.6 1/2 394

TABLE 5C HLA-A*0201 binding 9-mer peptides derived from HIG2 posi- stratpositive tive SEQ posi- Binding donor well positive ID tion sequenceScore number number CTL line NO 8 YLLGVVLTL 836.253 1/1 114 13 VLTLLSIFV650.311 0/1 0/12 — 115 15 TLLSIFVRV 488.951 1/1 116 4 VLNLYLLGV 271.9481/1 117 9 LLGVVLTLL 83.527 0/1 0/12 — 118 22 RVMESLEGL 31.957 0/1 0/12 —119 6 NLYLLGVVL 28.027 0/1 0/12 — 120

TABLE 5D HLA-A*0201 binding 10-mer peptides derived from HIG2 posi-posi- strat tive tive SEQ posi- Binding donor well positive ID tionsequence Score number number CTL line NO 8 YLLGvVLTLL 836.253 1/1 121 12VVLTlLSIFV 210.538 — — — 122 29 GLLEsPSPGT 113.047 0/1 0/12 — 123 6NLYLlGVVLT 54.847 — — — 124 4 VLNLyLLGVV 14.495 0/1 0/12 — 125 15TLLSiFVRVM 13.174 0/1 0/12 — 126 18 SIFVrVMESL 12.248 0/1 0/12 — 127 14LTLLsIFVRV 11.545 — — — 128

TABLE 6A HLA-A*2402 binding 9-mer peptides derived from INHBB posi-posi- tive tive SEQ strat Binding donor well positive ID positionsequence Score number number CTL line NO 383 LYFDDEYNI 60 0/3 0/20 — 129238 LFERGERRL 30 0/3 1/19 0/1 130 7 RALGAACLL 12 0/3 0/21 — 131 388EYNIVKRDV 10.5 0/3 0/18 — 132 180 LYLKLLPYV 9 1/2 395 163 ISNEGNQNL 8.640/1 0/8 — 396 223 RSGWHTFPL 8 0/1 0/6 — 397 176 ASLWLYLKL 7.92 0/1 0/7 —398 338 AYLAGVPGS 7.5 0/1 1/7 0/1 399 213 NMVEKRVDL 7.2 0/1 0/8 — 400102 AMVTALRKL 6.6 0/1 0/8 — 401 250 VQCDSCQEL 6.336 0/1 0/8 — 402 369NSCCIPTKL 6.16 0/1 0/8 — 403 330 NYCEGSCPA 6 0/1 0/7 — 404 172 FVVQASLWL6 0/1 0/8 — 405 355 VNQYRMRGL 6 0/1 0/8 — 406 307 QFFIDFRLI 6 0/1 0/7 —407 14 LLLLAAGWL 6 — — — 408 306 QQFFIDFRL 5.6 0/1 0/6 — 409 170NLFVVQASL 5.6 0/1 0/7 — 410 327 YYGNYCEGS 5 0/1 1/8 0/1 411

TABLE 6B HLA-A*2402 binding 10-mer peptides derived from INHBB posi-posi- strat tive tive SEQ posi- Binding donor well positive ID tionsequence Score number number CTL line NO 180 LYLKLLPYVL 360 1/3 133 171LFVVQASLWL 30 — — — 134 305 RQQFFIDFRL 16.8 1/3 135 73 DFLEAVKRHI 12.60/3 4/20 0/4 136 7 RALGAACLLL 12 1/3 137 273 RPFVVVQARL 11.2 0/3 1/200/1 138 338 AYLAGVPGSA 10 0/3 2/20 0/2 139 169 QNLFvVQASL 8.4 0/1 1/60/1 412 249 DVQCdSCQEL 7.92 0/1 4/6 0/4 413 383 LYFDdEYNIV 7.2 0/1 0/6 —415 229 FPLTeAIQAL 7.2 0/1 1/6 0/1 416 299 RTNLcCRQQF 7.2 0/1 5/6 0/5417 101 AAMVtALRKL 6.6 0/1 2/6 0/2 418 368 VNSCcIPTKL 6.16 0/1 2/6 0/2419 13 CLLLlAAGWL 6 — — — 420 354 VVNQyRMRGL 6 0/1 0/6 — 421 150DGLAsSRVRL 6 0/1 2/6 0/2 422 293 GLECdGRTNL 6 0/1 0/6 423 330 NYCEgSCPAY6 0/1 1/6 0/1 424 176 ASLWlYLKLL 6 0/1 1/6 0/1 425 212 WNMVeKRVDL 6 1/1426 74 FLEAvKRHIL 6 0/1 2/6 0/2 427 331 YCEGsCPAYL 6 0/1 1/6 0/1 428 77AVKRhILSRL 5.6 0/1 1/6 0/1 429 175 QASLwLYLKL 5.28 0/1 2/6 0/2 430 326GYYGnYCEGS 5 0/1 1/6 0/1 431 159 LYFFiSNEGN 5 0/1 4/6 0/4 432 327YYGNyCEGSC 5 0/1 1/6 0/1 433

TABLE 6C HLA-A*0201 binding 9-mer peptides derived from INHBB posi-posi- tive tive SEQ strat Binding donor well positive ID positionsequence Score number number CTL line NO 177 SLWLYLKLL 407.808 0/1 0/8140 14 LLLLAAGWL 96.074 — — — 141 170 NLFVVQASL 79.041 0/1 0/8 142 213NMVEKRVDL 63.256 0/1 0/8 143 172 FVVQASLWL 47.291 0/1 0/8 144 306QQFFIDFRL 46.48 0/1 0/8 145 281 RLGDSRHRI 42.774 0/1 0/8 146 174VQASLWLYL 34.427 0/1 0/8 147 257 ELAVVPVFV 28.69 0/1 1/8 0/1 148 313RLIGWNDWI 28.116 0/1 1/8 0/1 149 139 RVSEIISFA 22.546 0/1 3/8 0/3 150151 GLASSRVRL 21.362 0/1 0/8 151 8 ALGAACLLL 21.362 0/1 1/8 0/1 152 250VQCDSCQEL 15.096 0/1 1/8 0/1 153

TABLE 6D Table 6D HLA-A*0201 binding 10-mer peptides derived from INHBBposi- posi- strat tive tive SEQ posi- Binding donor well positive IDtion sequence Score number number CTL line NO 179 WLYLKLLPYV 12951.1 0/11/8 0/1 154 301 NLCCRQQFFI 332.806 0/1 0/8 155 237 ALFERGERRL 64.814 0/10/8 156 382 MLYFDDEYNI 56.754 0/1 0/8 157 13 CLLLLAAGWL 56.514 — — — 1588 ALGAACLLLL 49.134 — — — 159 313 RLIGWNDWII 32.081 0/1 0/8 160 173VVQASLWLYL 29.711 0/1 2/8 0/2 161 256 QELAVVPVFV 27.521 0/1 0/8 162 162FISNEGNQNL 13.512 0/1 1/8 0/1 163 305 RQQFFIDFRL 12.562 0/1 0/8 164 362GLNPGTVNSC 11.426 0/1 0/7 165 85 RLQMRGRPNI 10.433 0/1 1/8 0/1 166 69RVDGDFLEAV 10.425 0/1 0/8 167

TABLE 7A HLA-A*2402 binding 9-mer peptides derived from KIF20A posi-posi- tive tive SEQ strat Binding donor well positive ID positionsequence Score number number CTL line NO 308 IYNELLYDL 432 0/2 0/14 —168 621 MYEEKLNIL 432 0/2 0/14 — 169 67 VYLRVRPLL 420 0/2 0/14 — 170 499KFSAIASQL 56 0/2 0/14 — 171 304 SFFEIYNEL 44.352 0/2 0/14 — 172 187IFNSLQGQL 36 0/2 0/14 — 173 305 FFEIYNELL 30 1/2 174 23 MFESTAADL 30 0/20/14 — 175 256 SFDSGIAGL 20 0/2 0/14 — 176 298 RFSIWISFF 20 — — — 177383 IFSIRILHL 20 1/2 178 647 KIEELEALL 17.28 0/2 0/14 — 179 625KLNILKESL 14.4 0/2 0/14 — 180 695 KLQQCKAEL 13.2 0/2 0/14 — 181 726FTIDVDKKL 11.088 0/2 0/14 — 182 688 QLQEVKAKL 11.088 0/2 0/14 — 183

TABLE 7B HLA-A*2402 binding 10-mer peptides derived from KIF20A posi-posi- strat tive tive SEQ posi- Binding donor well positive ID tionsequence Score number number CTL line NO 308 IYNElLYDLL 432 0/2 0/14 —184 182 RSLAlIFNSL 24.192 0/2 1/14 0/1 185 304 SFFEiYNELL 24 1/2 186 742RLLRtELQKL 15.84 0/2 0/14 — 187 739 KNIRlLRTEL 15.84 0/2 0/14 — 188 218RQEEmKKLSL 14.4 0/2 2/14 0/2 189 70 RVRPlLPSEL 12.672 0/2 0/14 — 190 871RILRsRRSPL 12 0/2 0/14 — 191 89 RIENvETLVL 12 0/2 1/14 0/1 192 364KNQSfASTHL 12 0/2 0/14 — 193 66 KVYLrVRPLL 11.2 1/2 194 60 DSMEkVKVYL10.08 0/2 0/14 — 195

TABLE 8A HLA-A*2402 binding 9-mer peptides derived from KNTC2 posi-posi- tive tive SEQ strat Binding donor well positive ID positionsequence Score number number CTL line NO 309 KYQAYMSNL 600 1/3 196 457VYVPLKELL 432 0/3 0/18 — 197 414 EYHKLARKL 264 0/3 0/18 — 198 139SYELPDTKF 165 0/3 0/18 — 199 629 KYEKKATLI 150 0/3 0/18 — 200 400KYARGKEAI 100 0/3 1/18 0/1 201 124 DFLKIFTFL 50.4 1/3 202 134 GFLCPSYEL33 0/3 0/18 — 203 257 LFNVDAFKL 33 0/3 0/18 — 204 242 SFDEMNAEL 26.4 0/30/18 — 205 128 IFTFLYGFL 24 0/3 0/18 — 206 146 KFEEEVPRI 18 0/3 1/18 0/1207 368 RINHERNEL 15.84 0/3 1/18 0/1 208 235 SFMSGADSF 15 0/3 0/18 — 209154 IFKDLGYPF 14.4 1/3 210 563 EYQLVVQTT 12.6 0/3 0/18 — 211 474KALNKKMGL 12 0/3 1/18 0/1 212 150 EVPRIFKDL 10.08 1/3 213

TABLE 8B HLA-A*2402 binding 10-mer peptides derived from KNTC2 posi-posi- strat tive tive SEQ posi- Binding donor well positive ID tionsequence Score number number CTL line NO 452 KYRAQVYVPL 560 2/3 214 610EYEECMSEDL 360 0/3 1/18 0/1 215 360 KYSVADIERI 100 0/3 0/18 — 216 227DYTIKCYESF 100 1/3 217 146 KFEEEVPRIF 50.4 0/3 0/18 — 218 90 AFIQQCIRQL30 0/3 0/18 — 219 20 RSQDVNKQGL 17.28 0/3 1/18 0/1 220 501 RTLKEEVQKL15.84 0/3 0/18 — 221 403 RGKEAIETQL 13.44 0/3 1/18 0/1 222 273RALNEQIARL 12 1/3 223 563 EYQLVVQTTT 10.5 0/3 3/22 0/3 224 467ETEEEINKAL 10.08 0/3 1/22 0/1 225 541 LLESTVNQGL 10.08 0/3 1/22 0/1 226

TABLE 9A HLA-A*0201 binding 9-mer peptides derived from TTK posi- posi-tive tive SEQ strat Binding donor well positive ID position sequenceScore number number CTL line NO 462 YMSCFRTPV 878.055 1/1 227 547KQIYAIKYV 312.218 1/1 228 630 NMLEAVHTI 262.897 0/1 1/8 0/1 229 278LLNSPDCDV 118.238 0/1 1/8 0/1 230 498 ILATPLQNL 83.527 0/1 0/8 — 231 811YVLGQLVGL 73.172 0/1 0/8 — 232 719 SLGCILYYM 62.845 1/2 233 670QMQPDTTSV 50.232 0/1 0/8 — 234 804 GTTEEMKYV 50.102 0/1 0/8 — 235 654LIVDGMLKL 47.088 0/1 1/8 0/1 236 363 SLLAKLEET 31.074 0/1 0/8 — 237 790YVQIQTHPV 27.995 0/1 0/8 — 238 785 LLAHPYVQI 26.604 0/1 0/8 — 239 86KLIGRYSQA 26.082 0/1 0/8 — 240 186 NLNLQKKQL 21.362 0/1 0/8 — 241 671MQPDTTSVV 20.152 0/1 0/8 — 242 577 KLQQHSDKI 17.892 0/1 0/8 — 243 142FAFVHISFA 14.856 0/1 0/8 — 244 322 CELRNLKSV 11.509 0/1 0/8 — 245 824SILKAAKTL 10.868 0/1 0/8 — 246

TABLE 9B HLA-A*0201 binding 10-mer peptides derived from TTK posi- posi-strat tive tive SEQ posi- Binding donor well positive ID tion sequenceScore number number CTL line NO 68 LLLKLEKNSV 437.482 0/1 0/8 — 247 277NLLNSPDCDV 257.342 0/1 0/8 — 248 653 FLIVDGMLKL 226.014 0/1 0/8 — 249423 TTFEQPVFSV 195.487 0/1 0/8 — 250 542 VLNEKKQIYA 190.448 0/1 0/8 —251 658 GMLKLIDFGI 161.697 0/1 0/8 — 252 194 LLSEEEKKNL 148.896 0/1 0/8— 253 462 YMSCFRTPVV 94.738 1/1 254 57 MMANNPEDWL 70.685 0/1 0/8 — 255600 MVMECGNIDL 48.205 0/1 0/8 — 256 689 YMPPEAIKDM 37.961 0/1 0/8 — 25786 KLIGRYSQAI 36.515 0/1 0/8 — 258 669 NQMQPDTTSV 26.092 0/1 1/8 0/1 259497 QILATPLQNL 24.997 0/1 0/8 — 260 654 LIVDGMLKLI 22.997 0/1 0/8 — 261186 NLNLQKKQLL 21.362 0/1 1/8 0/1 262 670 QMQPDTTSVV 20.595 0/1 0/8 —263 803 KGTTEEMKYV 20.102 0/1 0/8 — 264 11 LTIDSIMNKV 15.486 0/1 0/8 —265 577 KLQQHSDKII 14.971 0/1 0/8 — 266

TABLE 10A HLA-A*0201 binding 9-mer peptides derived from URLC10 posi-posi- tive tive SEQ strat Binding donor well positive ID positionsequence Score number number CTL line NO 131 KIFPRFFMV 1364.78 0/1 0/8 —267 204 GLWLAILLL 407.808 0/1 0/8 — 268 65 LLVVALPRV 271.948 0/1 0/8 —269 60 ALLALLLVV 242.674 — — — 270 206 WLAILLLLA 52.561 1/1 271 212LLASIAAGL 36.316 1/1 272 210 LLLLASIAA 31.249 0/1 0/8 — 273 137FMVAKQCSA 16.505 0/1 2/8 0/2 274 58 TMALLALLL 15.428 0/1 2/8 0/2 275 59MALLALLLV 13.975 0/1 2/8 0/2 276 209 ILLLLASIA 12.812 0/1 0/8 — 434 208AILLLLASI 12.208 — — — 277 69 ALPRVWTDA 8.446 0/1 0/8 — 278 197SMGESCGGL 8.223 0/1 0/8 — 279 61 LLALLLVVA 7.964 — — — 280 67 VVALPRVWT6.097 0/1 0/8 — 281 72 RVWTDANLT 5.412 0/1 0/8 — 282 160 FLLEEPMPF 5.20/1 1/8 0/1 283 62 LALLLVVAL 4.292 0/1 0/8 — 284 57 GTMALLALL 2.525 0/11/8 0/1 285

TABLE 10B HLA-A*0201 binding 10-mer peptides derived from URLC10 posi-posi- strat tive tive SEQ posi- Binding donor well positive ID tionsequence Score number number CTL line NO 64 LLLVVALPRV 1006.21 0/1 0/8 —286 204 GLWLAILLLL 407.808 0/1 1/8 0/1 287 211 LLLASIAAGL 134.369 1/1288 258 TMALLALLLV 115.534 — — — 289 61 LLALLLVVAL 83.527 — — — 290 160FLLEEPMPFF 65.782 0/1 0/8 — 291 209 ILLLLASIAA 31.249 0/1 0/8 — 292 131KIFPRFFMVA 26.186 0/1 0/8 — 293 60 ALLALLLVVA 17.334 — — — 294 66LVVALPRVWT 6.097 0/1 0/8 — 295 59 MALLALLLVV 5.73 — — — 296 2 RLQRPRQAPA4.968 0/1 1/8 0/1 297 112 CQNPRRCKWT 4.156 0/1 0/8 — 298 72 RVWTDANLTA3.608 0/1 0/8 — 299 53 WAPLGTMALL 3.139 0/1 0/8 — 300 121 TEPYCVIAAV3.111 0/1 0/8 — 301 162 LEEPMPFFYL 2.739 0/1 1/8 0/1 302 181 LEGPPINSSV2.299 0/1 2/8 0/2 303 170 YLKCCKIRYC 2.024 0/1 0/8 — 304 130 VKIFPRFFMV1.81 0/1 0/8 — 305Stimulation of the T Cells Using the Predicted Peptides from CDH3Restricted with HLA-A*2402 and Establishment for CTL Lines Stimulatedwith CDH3 Derived Peptides

CTLs for those peptides derived from CDH3 were generated according tothe protocols set forth in “Materials and Methods” section above.Resulting that CTLs having detectable specific CTL activity, asdetermined by IFN-gamma ELISPOT assay, are shown in FIG. 1. Inparticular, CDH3-A24-9-513 (SEQ ID NO: 19), CDH3-A24-9-406 (SEQ ID NO:22), CDH3-A24-10-807 (SEQ ID NO: 30), CDH3-A24-10-332 (SEQ ID NO: 34),CDH3-A24-10-655 (SEQ ID NO: 344) and CDH3-A24-10-470 (SEQ ID NO: 358)demonstrated potent IFN-gamma production as compared to the control byIFN-gamma ELISPOT assay, and the cells in the positive well number #5stimulated with SEQ ID NO: 19, #2 with SEQ ID NO: 22, #5 with SEQ ID NO:30, #4 with SEQ ID NO: 34, #1 with SEQ ID NO: 344 and #4 with SEQ ID NO:358 were expanded and CTL lines were established. Those CTL lines havinghigher specific CTL activities against the peptide-pulsed target ascompared to the activities against target without peptide pulse weredetermined by ELISA. Results are shown in FIG. 1. While, other peptidesshown in table 2 could not establish the CTL lines despite possiblebinding activity with HLA-A*2402. For example, the typical negativepeptide (CDH3-A24-10-248) were shown in FIG. 1a . In this invention, thepeptides which could establish CTL line were selected as potent CTLstimulation peptide.

Establishment for CTL Clones Stimulated with CDH3 Derived Peptides

Furthermore, the limiting dilution from these CTL lines was performedaccording to the protocols set forth in the “Materials and Methods”section above. The establishment of CTL clones from CDH3-A24-10-807 (SEQID NO: 30) #5 and CDH3-A24-10-655 (SEQ ID NO: 344) #1 CTL line are shownin FIG. 1f and g . CTL clones had potent and specific CTL activitiesagainst the peptide-pulsed target as compared to the activities againsttarget without peptide pulse.

Specific CTL Activity Against the Target Cells Expressing CDH3 andHLA-A*2402

The established CTL line raised against these peptides were examined fortheir ability to recognize the target cells expressing CDH3 andHLA-A*2402. Specific CTL activity against COS7 transfected with bothfull length CDH3 gene and the HLA-A*2402 molecule, which serves as aspecific model for the target cells endogenously express CDH3 andHLA-A*2402, was tested using as effector cells the CTL lines raised byCDH3-A24-10-807 (SEQ ID NO: 30) and CDH3-A24-10-655 (SEQ ID NO: 344).COS7 transfected with full length CDH3 but not HLA-A*2402 and COS7transfected with HLA-A*2402 but not full length CDH3 were prepared ascontrols. The CTL clones demonstrated the highest specific CTL activityagainst COS7 that was transfected with both CDH3 and HLA-A2402 (Figureif and g).

These results clearly demonstrate that CDH3-A24-10-807 (SEQ ID NO: 30)and CDH3-A24-10-655 (SEQ ID NO: 344) are naturally expressed on thetarget cell surface with HLA-A2402 molecule and recognize CTL.Furthermore, these peptides are epitope peptides, which may serve ascancer vaccines targeting CDH3 expressed tumors.

Stimulation of the T Cells Using the Predicted Peptides from EPHA4Restricted with HLA-A*2402 or HLA-A*0201, and Establishment for CTLLines Stimulated with EPHA4 Derived Peptides

CTLs for those peptides derived from EphA4 were generated by IFN-gammaELISPOT assay. Resulting that CTLs having detectable specific CTLactivity, as determined by IFN-gamma ELISPOT assay, are shown in FIG. 2.In particular, EphA4-A24-9-453 (SEQ ID NO: 41), EphA4-A24-9-5 (SEQ IDNO: 44), EphA4-A24-9-869 (SEQ ID NO: 46), EphA4-A24-9-420 (SEQ ID NO:48), EphA4-A24-10-24 (SEQ ID NO: 78), EphA4-A02-9-501 (SEQ ID NO: 376)and EphA4-A02-9-165 (SEQ ID NO: 379) demonstrated potent IFN-gammaproduction by IFN-gamma ELISPOT assay, and the cells in the positivewell number #3 stimulated with EphA4-A24-9-453 (SEQ ID NO: 41), #2 withEphA4-A24-9-5 (SEQ ID NO: 44), #5 with EphA4-A24-9-869 (SEQ ID NO: 46),#6 with EphA4-A24-9-420 (SEQ ID NO: 48), #4 with EphA4-A24-10-24 (SEQ IDNO: 78), #8 with EphA4-A02-9-501 (SEQ ID NO: 376) and #3 withEphA4-A02-9-165 (SEQ ID NO: 379) were expanded and CTL lines wereestablished. Those CTL lines having higher specific CTL activitiesagainst the peptide-pulsed target as compared to the activities againsttarget without peptide pulse were determined by ELISA. Especially, CTLlines stimulated with EphA4-A02-9-501 (SEQ ID NO: 376) andEphA4-A02-9-165 (SEQ ID NO: 379) were tested by 51Cr-release assayaccording to the protocols set forth in the “Materials and Methods”section above. Results are shown in FIG. 2a-h . While, other peptidesshown in table 3 could not establish the CTL lines despite possiblebinding activity with HLA-A*2402 or HLA-A*0201. For example, the typicalnegative peptide (EphA4-A24-9-384) were shown in FIG. 2a . In thisinvention, the peptides which could establish CTL line were selected aspotent CTL stimulation peptides.

Stimulation of the T Cells Using the Predicted Peptides from ECT2Restricted with HLA-A*2402, and Establishment for CTL Lines Stimulatedwith ECT2 Derived Peptides

CTLs for those peptides derived from ECT2 were generated according tothe protocols set forth in the “Materials and Methods” section above.Resulting CTLs having detectable specific CTL activity as determined byan IFN-gamma ELISPOT assay are shown in FIG. 3. In particular,ECT2-A24-9-515 (SEQ ID NO: 80), ECT2-A24-10-40 (SEQ ID NO: 100) andECT2-A24-10-101 (SEQ ID NO: 101) showed potent IFN-gamma production, andthe cells in the positive well number #7 stimulated with ECT2-A24-9-515(SEQ ID NO: 80), #2 with ECT2-A24-10-40 (SEQ ID NO: 100) and #1 withECT2-A24-10-101 (SEQ ID NO: 101) were expanded and CTL lines wereestablished. Those CTL lines having higher specific CTL activitiesagainst the peptide-pulsed target as compared to the activities againsttarget without peptide pulse were determined by ELISA. Results are shownin FIG. 3a-d . While, other peptides shown in table 4 could notestablish the CTL lines despite possible binding activity withHLA-A*2402. For example, the typical negative peptide (ECT2-A24-10-322,ECT2-A24-9-657 and ECT2-A24-10-811) were shown in FIG. 2a . In thisinvention, the peptides which could establish CTL line were selected aspotent CTL stimulation peptide.

Establishment for CTL Clones Stimulated with ECT2 Derived Peptides

Furthermore, the limiting dilution from these CTL lines was performedaccording to the protocols set forth in the “Materials and Methods”section above. The establishment of CTL clones from ECT2-A24-10-40 (SEQID NO: 100) #2 CTL line are shown in FIG. 3c . CTL clones had potent andspecific CTL activities against the peptide-pulsed target as compared tothe activities against target without peptide pulse.

Specific CTL Activity Against the Target Cells Expressing ECT2 andHLA-A*2402

The established CTL line raised against these peptides were examined fortheir ability to recognize the target cells expressing ECT2 andHLA-A*2402. Specific CTL activity against COS7 transfected with bothfull length ECT2 gene and the HLA-A*2402 molecule, which serves as aspecific model for the target cells endogenously express ECT2 andHLA-A*2402, was tested using as effector cells the CTL clone raised byECT2-A24-10-40 (SEQ ID NO: 100) and the CTL line raised byECT2-A24-10-101 (SEQ ID NO: 101). COS7 transfected with full length ECT2but not HLA-A*2402 and COS7 transfected with HLA-A*2402 but not fulllength ECT2 (replaced other gene e.g. URLC10 or INHBB) were prepared ascontrols. The CTL line demonstrating the highest specific CTL activityagainst COS7 that was transfected with both ECT2 and HLA-A2402 (FIGS. 3cand d ).

These results clearly demonstrate that ECT2-A24-10-40 (SEQ ID NO: 100)and ECT2-A24-10-101 (SEQ ID NO: 101) are naturally expressed on thetarget cell surface with HLA-A2402 molecule and recognize CTL.Furthermore, these peptides are epitope peptides, which may serve ascancer vaccines targeting ECT2 expressed tumors.

Cytotoxic Activity Against Cancer Cell Line Endogenously ExpressingHLA-A*2402 and ECT2

Furthermore, Cytotoxic activity was examined by cytotoxicity assayaccording to the protocols set forth in the “Materials and Methods”section above. As a result, as shown in FIG. 3b , CTL clone stimulatedwith ECT2-A24-9-515 (SEQ ID NO: 80) showed remarkably high cytotoxiceffect towards HLA-A24-positive and ECT-positive cancer cell lines TE6,compared to that towards HLA-A24-negative and ECT-positive cancer celllines TE5.

Stimulation of the T Cells Using the Predicted Peptides from HIG2Restricted with HLA-A*2402 or HLA-A*0201, and Establishment for CTLLines Stimulated with HIG2 Derived Peptides

CTLs for those peptides derived from HIG2 were generated according tothe protocols set forth in the “Materials and Methods” section above.Resulting CTLs having detectable specific CTL activity as determined byan IFN-gamma ELISPOT assay are shown in FIG. 4. In particular,HIG2-A24-9-19 (SEQ ID NO: 110), HIG2-A24-9-22 (SEQ ID NO: 111),HIG2-A24-9-8 (SEQ ID NO: 387), HIG2-A24-10-7 (SEQ ID NO: 112),HIG2-A24-10-18 (SEQ ID NO: 394), HIG2-A02-9-8 (SEQ ID NO: 114),HIG2-A02-9-15 (SEQ ID NO: 116), HIG2-A02-9-4 (SEQ ID NO: 117) andHIG2-A02-10-8 (SEQ ID NO: 121) demonstrated potent IFN-gamma productionby IFN-gamma ELISPOT assay, and the cells in the positive well number #6stimulated with HIG2-A24-9-19 (SEQ ID NO: 110), #7 with HIG2-A24-9-22(SEQ ID NO: 111), #5 with HIG2-A24-9-8 (SEQ ID NO: 387), #1 withHIG2-A24-10-7 (SEQ ID NO: 112), #7 with HIG2-A24-10-18 (SEQ ID NO: 394),#10 with HIG2-A02-9-8 (SEQ ID NO: 114), #10 with HIG2-A02-9-15 (SEQ IDNO: 116), #10 with HIG2-A02-9-4 (SEQ ID NO: 117) and #9 withHIG2-A02-10-8 (SEQ ID NO: 121) were expanded and CTL lines wereestablished. Those CTL lines having higher specific CTL activitiesagainst the peptide-pulsed target as compared to the activities againsttarget without peptide pulse were determined by ELISA. Results are shownin FIG. 4a-j . While, other peptides shown in table 5 could notestablish the CTL lines despite possible binding activity withHLA-A*2402. For example, the typical negative peptide (HIG2-A24-9-7)were shown in FIG. 4a . In this invention, the peptides which couldestablish CTL line were selected as potent CTL stimulation peptide.

Establishment for CTL Clones Stimulated with HIG2 Derived Peptides

Furthermore, the limiting dilution from these CTL lines was performedaccording to the protocols set forth in the “Materials and Methods”section above. The establishment of CTL clones from HIG2-A24-9-22 (SEQID NO: 111) #7 CTL line, HIG2-A24-9-8 (SEQ ID NO: 387) #5 CTL line,HIG2-A24-10-7 (SEQ ID NO: 112) #1 CTL line, HIG2-A24-10-18 (SEQ ID NO:394) #7 CTL line and HIG2-A02-9-4 (SEQ ID NO: 117) #10 CTL line areshown in FIGS. 4 c, e, f, g and i. CTL clones had potent and specificCTL activities against the peptide-pulsed target as compared to theactivities against target without peptide pulse.

Specific CTL Activity Against the Target Cells Expressing HIG2 andHLA-A*0201

The established CTL line raised against these peptides were examined fortheir ability to recognize the target cells expressing HIG2 andHLA-A*0201. Specific CTL activity against 293T or COS7 transfected withboth full length HIG2 gene and the HLA-A*0201 molecule, which serves asa specific model for the target cells endogenously express HIG2 andHLA-A*0201, was tested using as effector cells the CTL lines raised byHIG2-A02-9-8 (SEQ ID NO: 114), HIG2-A02-9-15 (SEQ ID NO: 116) and theCTL clone raised by HIG2-A02-9-4 (SEQ ID NO: 117). 293T or COS7transfected with full length ECT2 but not HLA-A*0201 and 293T or COS7transfected with HLA-A*0201 but not full length ECT2 (or replaced othergene e.g. FoxP3 or TTK) were prepared as controls. The CTL linedemonstrating the highest specific CTL activity against 293T or COS7that was transfected with both ECT2 and HLA-A*0201 (FIGS. 4e, h and i ).

These results clearly demonstrate that HIG2-A02-9-8 (SEQ ID NO: 114),HIG2-A02-9-15 (SEQ ID NO: 116) and HIG2-A02-9-4 (SEQ ID NO: 117) arenaturally expressed on the target cell surface with HLA-A2402 orHLA-A0201 molecule and recognize CTL. Furthermore, these peptides areepitope peptides, which may serve as cancer vaccines targeting HIG2expressed tumors.

Cytotoxic Activity Against Cancer Cell Line Endogenously ExpressingHLA-A*0201 and HIG2

Furthermore, Cytotoxic activity was examined by cytotoxicity assayaccording to the protocols set forth in the “Materials and Methods”section above. As a result, as shown in FIG. 4i , CTL clone stimulatedwith HIG2-A02-9-4 (SEQ ID NO: 117) showed remarkably high cytotoxiceffect towards HLA-A02-positive and HIG2-positive cancer cell linesCAki-1, compared to that towards HLA-A02-negative and HIG2-positivecancer cell lines A498.

Stimulation of the T Cells Using the Predicted Peptides from INHBBRestricted with HLA-A*2402 or HLA-A*0201, and Establishment for CTLLines Stimulated with INHBB Derived Peptides

CTLs for those peptides derived from INHBB were generated according tothe protocols set forth in the “Materials and Methods” section above.Resulting CTLs having detectable specific CTL activity as determined byan IFN-gamma ELISPOT assay are shown in FIG. 5. In particular,INHBB-A24-9-180 (SEQ ID NO: 395), INHBB-A24-10-180 (SEQ ID NO: 133),INHBB-A24-10-305 (SEQ ID NO: 135), INHBB-A24-10-7 (SEQ ID NO: 137) andINHBB-A24-10-212 (SEQ ID NO: 426) demonstrated potent IFN-gammaproduction by IFN-gamma ELISPOT assay, and the cells in the positivewell number #7 stimulated with INHBB-A24-9-180 (SEQ ID NO: 395), #3 withINHBB-A24-10-180 (SEQ ID NO: 133), #2 with INHBB-A24-10-305 (SEQ ID NO:135), #8 and #2 with INHBB-A24-10-7 (SEQ ID NO: 137) and #1 withINHBB-A24-10-212 (SEQ ID NO: 426) were expanded and CTL lines wereestablished. Those CTL lines having higher specific CTL activitiesagainst the peptide-pulsed target as compared to the activities againsttarget without peptide pulse were determined by ELISA. Results are shownin FIG. 5b-e . While, other peptides shown in table 6 could notestablish the CTL lines despite possible binding activity withHLA-A*2402 and HLA*0201. For example, the typical negative peptide(INHBB-A24-9-238) were shown in FIG. 5a . In this invention, thepeptides which could establish CTL line were selected as potent CTLstimulation peptide.

Establishment for CTL Clones Stimulated with INHBB Derived Peptides

Furthermore, the limiting dilution from these CTL lines was performedaccording to the protocols set forth in the “Materials and Methods”section above. The establishment of CTL clones from INHBB-A24-9-180 (SEQID NO: 395) #7 CTL line, and INHBB-A24-10-305 (SEQ ID NO: 135) #2 CTLline are shown in FIGS. 5b and d . CTL clones had potent and specificCTL activities against the peptide-pulsed target as compared to theactivities against target without peptide pulse.

Specific CTL Activity Against the Target Cells Expressing INHBB andHLA-A*2402

The established CTL line raised against these peptides were examined fortheir ability to recognize the target cells expressing INHBB andHLA-A*2402. Specific CTL activity against 293T transfected with bothfull length INHBB gene and the HLA-A*2402 molecule, which serves as aspecific model for the target cells endogenously express INHBB andHLA-A*2402, was tested using as effector cells the CTL lines raised byINHBB-A24-10-180 (SEQ ID NO: 133) and INHBB-A24-10-7 (SEQ ID NO: 137)and the CTL clone raised by INHBB-A24-10-305 (SEQ ID NO: 135). 293Ttransfected with full length INHBB but not HLA-A*2402 and 293Ttransfected with HLA-A*2402 but not full length INHBB were prepared ascontrols. The CTL line demonstrating the highest specific CTL activityagainst 293T was that transfected with both INHBB and HLA-A*2402 (FIGS.5c, d and e ).

These results clearly demonstrate that INHBB-A24-10-305 (SEQ ID NO:135), INHBB-A24-10-180 (SEQ ID NO: 133) and INHBB-A24-10-7 (SEQ ID NO:137) are naturally expressed on the target cell surface with HLA-A2402molecule and recognize CTL.

Furthermore, these peptides are epitope peptides, which may serve ascancer vaccines targeting INHBB expressed tumors.

Cytotoxic Activity Against Cancer Cell Line Endogenously ExpressingHLA-A*2402 and INHBB

Furthermore, Cytotoxic activity was performed by cytotoxicity assayaccording to the protocols set forth in the “Materials and Methods”section above. As a result, as shown in FIG. 5b , CTL clone stimulatedwith INHBB-A24-9-180 (SEQ ID NO: 395) showed remarkably high cytotoxiceffect towards HLA-A24-positive and INHBB-positive cancer cell linesMIAPaca2, compared to that towards HLA-A24-negative and INHBB-positivecancer cell lines CAki-2.

Stimulation of the T Cells Using the Predicted Peptides from KIF20ARestricted with HLA-A*2402, and Establishment for CTL Lines Stimulatedwith KIF20A Derived Peptides

CTLs for those peptides derived from KIF20A were generated according tothe protocols set forth in the “Materials and Methods” section above.Resulting CTLs having detectable specific CTL activity as determined byan IFN-gamma ELISPOT assay are shown in FIG. 6. In particular,KIF20A-A24-9-305 (SEQ ID NO: 174), KIF20A-A24-9-383 (SEQ ID NO: 178),KIF20A-A24-10-304 (SEQ ID NO: 186) and KIF20A-A24-10-66 (SEQ ID NO: 194)demonstrated potent IFN-gamma production by IFN-gamma ELISPOT assay, andthe cells in the positive well number #2 stimulated withKIF20A-A24-9-305 (SEQ ID NO: 174), #3 with KIF20A-A24-9-383 (SEQ ID NO:178), #5 with KIF20A-A24-10-304 (SEQ ID NO: 186) and #6 withKIF20A-A24-10-66 (SEQ ID NO: 194) were expanded and CTL lines wereestablished. Those CTL lines having higher specific CTL activitiesagainst the peptide-pulsed target as compared to the activities againsttarget without peptide pulse were determined by ELISA. Results are shownin FIG. 6a-e . While, other peptides shown in table 7 could notestablish the CTL lines despite possible binding activity withHLA-A*2402. For example, the typical negative peptide (KIF20A-A24-9-647and KIF20A-A24-10-182) were shown in FIG. 6a . In this invention, thepeptides which could establish CTL line were selected as potent CTLstimulation peptide.

Establishment for CTL Clones Stimulated with KIF20A Derived Peptides

Furthermore, the limiting dilution from these CTL lines was performedaccording to the protocols set forth in the “Materials and Methods”section above. The establishment of CTL clones from KIF20A-A24-9-305(SEQ ID NO: 174) #2 CTL line, KIF20A-A24-10-304 (SEQ ID NO: 186) #5 CTLline and KIF20A-A24-10-66 (SEQ ID NO: 194) #6 CTL line are shown inFIGS. 6b, d and e . CTL clones had potent and specific CTL activitiesagainst the peptide-pulsed target as compared to the activities againsttarget without peptide pulse.

Specific CTL Activity Against the Target Cells Expressing KIF20A andHLA-A*2402

The established CTL line raised against these peptides were examined fortheir ability to recognize the target cells expressing KIF20A andHLA-A*2402. Specific CTL activity against COS7 transfected with bothfull length KIF20A gene and the HLA-A*2402 molecule and A24-LCLtransfected by electroporation with full length KIF20A gene, which serveas a specific model for the target cells endogenously express KIF20A andHLA-A*2402, was tested using as effector cells the CTL lines raised byKIF20A-A24-9-383 (SEQ ID NO: 178) and KIF20A-A24-10-304 (SEQ ID NO: 186)and the CTL clone raised by KIF20A-A24-10-66 (SEQ ID NO: 194). COS7transfected with full length KIF20A but not HLA-A*2402 and COS7transfected with HLA-A*2402 but not full length KIF20A (or replaced fulllength URLC10 gene), COS7 transfected with HLA-A*2402 and pulsed withKIF20A-10-308, and A24-LCL transfected with mock vector were prepared ascontrols. The CTL line demonstrated the highest specific CTL activityagainst COS7 that was transfected with both KIF20A and HLA-A*2402 (FIGS.6b, c and d ). Alternatively, the CTL line stimulated withKIF20A-A24-10-304 (SEQ ID NO: 186) demonstrated against A24-LCLtransfected with KIF20A.

These results clearly demonstrate that KIF20A-A24-9-383 (SEQ ID NO:178), KIF20A-A24-10-304 (SEQ ID NO: 186) and KIF20A-A24-10-66 (SEQ IDNO: 194) is naturally expressed on the target cell surface withHLA-A2402 molecule and recognize CTL. Furthermore, these peptides areepitope peptides, which may serve as cancer vaccines targeting KIF20Aexpressed tumors.

Cytotoxic Activity Against Cancer Cell Line Endogenously ExpressingHLA-A*2402 and KIF20A

Furthermore, Cytotoxic activity was examined by cytotoxicity assayaccording to the protocols set forth in the “Materials and Methods”section above. As a result, as shown in FIGS. 6b and e , CTL clonestimulated with KIF20A-A24-9-305 (SEQ ID NO: 174) or KIF20A-A24-10-304(SEQ ID NO: 186) showed remarkably high cytotoxic effect towardsHLA-A24-positive and KIF20A-positive cancer cell lines PK45P or MIAPaca2respectively, compared to that towards HLA-A24-negative andKIF20A-positive cancer cell lines PK59.

Stimulation of the T Cells Using the Predicted Peptides from KNTC2Restricted with HLA-A*2402, and Establishment for CTL Lines Stimulatedwith KNTC2 Derived Peptides

CTLs for those peptides derived from KNTC2 were generated according tothe protocols set forth in the “Materials and Methods” section above.Resulting CTLs having detectable specific CTL activity as determined byan IFN-gamma ELISPOT assay are shown in FIG. 7. In particular,KNTC2-A24-9-309 (SEQ ID NO: 196), KNTC2-A24-9-124 (SEQ ID NO: 202),KNTC2-A24-9-154 (SEQ ID NO: 210), KNTC2-A24-9-150 (SEQ ID NO: 213),KNTC2-A24-10-452 (SEQ ID NO: 214), KNTC2-A24-10-227 (SEQ ID NO: 217) andKNTC2-A24-10-273 (SEQ ID NO: 223) demonstrated potent IFN-gammaproduction by IFN-gamma ELISPOT assay, and the cells in the positivewell number #8 stimulated with KNTC2-A24-9-309 (SEQ ID NO: 196), #5 withKNTC2-A24-9-124 (SEQ ID NO: 202), #5 with KNTC2-A24-9-154 (SEQ ID NO:210), #7 with KNTC2-A24-9-150 (SEQ ID NO: 213), #4 and #5 withKNTC2-A24-10-452 (SEQ ID NO: 214), #1 with KNTC2-A24-10-227 (SEQ ID NO:217) and #8 with KNTC2-A24-10-273 (SEQ ID NO: 223) were expanded and CTLlines were established. Those CTL lines having higher specific CTLactivities against the peptide-pulsed target as compared to theactivities against target without peptide pulse were determined byELISA. Results are shown in FIG. 7a-h . While, other peptides shown intable 8 could not establish the CTL lines despite possible bindingactivity with HLA-A*2402. For example, the typical negative peptide(KNTC2-A24-10-610) were shown in FIG. 7a . In this invention, thepeptides which could establish CTL line were selected as potent CTLstimulation peptide.

Establishment for CTL Clones Stimulated with KNTC2 Derived Peptides

Furthermore, the limiting dilution from these CTL lines was performedaccording to the protocols set forth in the “Materials and Methods”section above. The establishment of CTL clones from KNTC2-A24-9-154 (SEQID NO: 210) #5 CTL line and KNTC2-A24-10-452 (SEQ ID NO: 214) #5 CTLline are shown in FIGS. 7d and f . CTL clones had potent and specificCTL activities against the peptide-pulsed target as compared to theactivities against target without peptide pulse.

Specific CTL Activity Against the Target Cells Expressing KNTC2 andHLA-A*2402

The established CTL line raised against these peptides were examined fortheir ability to recognize the target cells expressing KNTC2 andHLA-A*2402. Specific CTL activity against HEK293 transfected with bothfull length KNTC2 gene and the HLA-A*2402 molecule which serves as aspecific model for the target cells endogenously express KNTC2 andHLA-A*2402, was tested using as effector cells the CTL clones raised byKNTC2-A24-10-452 (SEQ ID NO: 214). HEK293 transfected with full lengthKNTC2 but not HLA-A*2402, HEK293 transfected with HLA-A*2402 but notfull length KNTC2 and HEK293 transfected with HLA-A*2402 and pulsed withKNTC2-9-309 were prepared as controls. The CTL line demonstrating thehighest specific CTL activity against HEK293 was that transfected withboth KNTC2 and HLA-A*2402 (FIG. 7f ).

These results clearly demonstrate that KNTC2-A24-10-452 (SEQ ID NO: 214)is naturally expressed on the target cell surface with HLA-A2402molecule and recognize CTL. Furthermore, these peptides are epitopepeptides, which may serve as cancer vaccines targeting KNTC2 expressedtumors.

Stimulation of the T Cells Using the Predicted Peptides from TTKRestricted with HLA-A*0201, and Establishment for CTL Lines Stimulatedwith TTK Derived Peptides

CTLs for those peptides derived from TTK were generated according to theprotocols set forth in the “Materials and Methods” section above.Resulting CTLs having detectable specific CTL activity as determined byan IFN-gamma ELISPOT assay are shown in FIG. 8. As depicted in FIG. 8b-d, TTK-A2-9-462 (SEQ ID NO: 227), TTK-A2-9-547 (SEQ ID NO: 228),TTK-A2-9-719 (SEQ ID NO: 233) and TTK-A2-10-462 (SEQ ID NO: 254)demonstrated potent IFN-gamma production by IFN-gamma ELISPOT assay, andthe cells in the positive well number #4 stimulated with TTK-A2-9-462(SEQ ID NO: 227), #2 with TTK-A2-9-547 (SEQ ID NO: 228), #1 withTTK-A2-9-719 (SEQ ID NO: 233) and #8 with TTK-A2-10-462 (SEQ ID NO: 254)were expanded. Those CTL lines having higher specific CTL activitiesagainst the peptide-pulsed target as compared to the activities againsttarget without peptide pulse were determined by ELISA. While, otherpeptides shown in table 9 could not establish the CTL lines despitepossible binding activity with HLA-A*0201. For example, the typicalnegative peptide (TTK-A2-9-278) were shown in FIG. 8a . In thisinvention, the peptides which could establish CTL line were selected aspotent CTL stimulation peptide.

Establishment for CTL Clones Stimulated with TTK Derived Peptides

Furthermore, the limiting dilution from these CTL lines was performedaccording to the protocols set forth in the “Materials and Methods”section above. The establishment of CTL clones from TTK-A2-9-462 (SEQ IDNO: 227) #4 CTL line, TTK-A2-9-547 (SEQ ID NO: 228) #2 CTL line,TTK-A2-9-719 (SEQ ID NO: 233) #1 CTL line and TTK-A2-10-462 (SEQ ID NO:254) #8 CTL line were shown in FIGS. 8 d, c, d and e. CTL clones hadpotent and specific CTL activities against the peptide-pulsed target ascompared to the activities against target without peptide pulse.

Specific CTL Activity Against the Target Cells Expressing TTK andHLA-A*0201

The established CTL clone raised against these peptides were examinedfor their ability to recognize the target cells endogenously expressingTTK and HLA-A*0201. Specific CTL activity against COS7 transfected withboth the full length TTK gene and the HLA-A*0201 molecule, which is aspecific model for the target cells endogenously express TTK andHLA-A*0201, was tested using as effector cells the CTL clones raised byTTK-A2-9-462 (SEQ ID NO: 227), TTK-A02-9-547 (SEQ ID NO: 228),TTK-A2-9-719 (SEQ ID NO: 233) and TTK-A2-10-462 (SEQ ID NO: 254). COS7transfected with full length TTK but HLA-A*0201, COS7 transfectedHLA-A*0201 but not full length of TTK (or replaced full length HIG2gene) and COS7 transfected with HLA-A*0201 and pulsed with differenttarget epitope peptide, were prepared as controls. The CTL Clone had thehighest specific CTL activity against COS7 that was transfected withboth TTK and HLA-A*0201 (FIGS. 8 b, c, d and e).

These results clearly demonstrate that TTK-A2-9-462 (SEQ ID NO: 227),TTK-A02-9-547 (SEQ ID NO: 228), TTK-A2-9-719 (SEQ ID NO: 233) andTTK-A02-10-462 (SEQ ID NO: 254) are naturally expressed on the targetcell surface with HLA-A2 (HLA-A02) molecule and recognize CTL.Furthermore, these peptides are epitope peptides, which may serve ascancer vaccines targeting TTK expressed tumors.

Stimulation of the T Cells Using the Predicted Peptides from URLC10Restricted with HLA-A*0201, and Establishment for CTL Lines Stimulatedwith URLC10 Derived Peptides

CTLs for those peptides derived from URLC10 were generated according tothe protocols set forth in the “Materials and Methods” section above.Resulting CTLs having detectable specific CTL activity as determined byIFN-gamma ELISPOT assay are shown in FIG. 9. As shown in FIG. 9b-d ,URLC-A2-9-206 (SEQ ID NO: 271), URLC-A2-9-212 (SEQ ID NO: 272) andURLC-A2-10-211 (SEQ ID NO: 288) demonstrated potent IFN-gamma productionby IFN-gamma ELISPOT assay, and the cells in the positive well number #7stimulated with URLC-A2-9-206 (SEQ ID NO: 271), #3 with URLC-A2-9-212(SEQ ID NO: 272) and #5 with URLC-A2-10-211 (SEQ ID NO: 288) wereexpanded. Those CTL lines having higher specific CTL activities againstthe peptide-pulsed target as compared to the activities against targetwithout peptide pulse were determined by ELISA. While, other peptidesshown in table 10 could not establish the CTL lines despite possiblebinding activity with HLA-A*0201. For example, the typical negativepeptide (URLC-A2-9-58) were shown in FIG. 9a . In this invention, thepeptide which could establish CTL line were selected as potent CTLstimulation peptide.

Specific CTL Activity Against the Target Cells Expressing URLC10 andHLA-A*0201

The established CTL line raised against these peptides were examined fortheir ability to recognize the target cells endogenously expressingURLC10 and HLA-A*0201. Specific CTL activity against COS7, Hek293 and293T transfected with both full length URLC10 gene and the HLA-A*0201molecule, which serves as a specific model for the target cellsendogenously express URLC10 and HLA-A*0201, was tested using as effectorcells the CTL line raised by URLC10-A02-10-211. COS7, Hek293 or 293Ttransfected with full length URLC10 but not HLA-A*0201 (replacedHLA-A*2402), COS7, Hek293 or 293T transfected with HLA-A*0201 but notfull length URLC10 and COS7 transfected with HLA-A*0201 and pulsed withdifferent target epitope peptide (URLC10-A02-10-64) were prepared ascontrols. The CTL line demonstrating the highest specific CTL activityagainst COS7, Hek293 or 293T was that transfected with both URLC10 andHLA-A*0201 (FIG. 9-2).

These results clearly demonstrate that URLC10-A02-10-211 is naturallyexpressed on the target cell surface with HLA-A*0201 molecule andrecognizes CTL. Furthermore, this peptide was epitope peptides, whichmay utilize cancer vaccine targeting URLC10 expressed tumors.

Homology Analysis of the Antigen Peptides

The CTL clones established against the following peptides showed potentspecific CTL activity.

-   -   CDH3-A24-9-513 (SEQ ID NO: 19),    -   CDH3-A24-9-406 (SEQ ID NO: 22),    -   CDH3-A24-10-807 (SEQ ID NO: 30),    -   CDH3-A24-10-332 (SEQ ID NO: 34),    -   CDH3-A24-10-655 (SEQ ID NO: 344),    -   CDH3-A24-10-470 (SEQ ID NO: 358),    -   EphA4-A24-9-453 (SEQ ID NO: 41),    -   EphA4-A24-9-5 (SEQ ID NO: 44),    -   EphA4-A24-9-869 (SEQ ID NO: 46),    -   EphA4-A24-9-420 (SEQ ID NO: 48),    -   EphA4-A24-10-24 (SEQ ID NO: 78),    -   EphA4-A02-9-501 (SEQ ID NO: 376),    -   EphA4-A02-9-165 (SEQ ID NO: 379),    -   HIG-A24-9-19 (SEQ ID NO: 110),    -   HIG-A24-9-22 (SEQ ID NO: 111),    -   HIG-A24-9-8 (SEQ ID NO: 387),    -   HIG-A24-10-7 (SEQ ID NO: 112),    -   HIG-A24-10-18 (SEQ ID NO: 394),    -   HIG-A02-9-8 (SEQ ID NO: 114),    -   HIG-A02-9-15 (SEQ ID NO: 116),    -   HIG-A02-9-4 (SEQ ID NO: 117),    -   HIG-A02-10-8 (SEQ ID NO: 121),    -   INHBB-A24-9-180 (SEQ ID NO: 395),    -   INHBB-A24-10-180 (SEQ ID NO: 133),    -   INHBB-A24-10-305 (SEQ ID NO: 135),    -   INHBB-A24-10-7 (SEQ ID NO: 137),    -   INHBB-A24-10-212 (SEQ ID NO: 426),    -   KIF20A-A24-9-305 (SEQ ID NO: 174),    -   KIF20A-A24-9-383 (SEQ ID NO: 178),    -   KIF20A-A24-10-304 (SEQ ID NO: 186),    -   KIF20A-A24-10-66 (SEQ ID NO: 194),    -   KNTC2-A24-9-309 (SEQ ID NO: 196),    -   KNTC2-A24-9-124 (SEQ ID NO: 202),    -   KNTC2-A24-9-154 (SEQ ID NO: 210),    -   KNTC2-A24-9-150 (SEQ ID NO: 213),    -   KNTC2-A24-10-452 (SEQ ID NO: 214),    -   KNTC2-A24-10-227 (SEQ ID NO: 217),    -   KNTC2-A24-10-273 (SEQ ID NO: 223),    -   TTK-A02-9-462 (SEQ ID NO: 227),    -   TTK-A02-9-547 (SEQ ID NO: 228),    -   TTK-A02-9-719 (SEQ ID NO: 233),    -   TTK-A02-10-462 (SEQ ID NO: 254),    -   URLC-A02-9-206 (SEQ ID NO: 271),    -   URLC-A02-9-212 (SEQ ID NO: 272) and    -   URLC-A02-10-211 (SEQ ID NO: 288)

This suggests that the sequences of SEQ ID NO: 19, 22, 30, 34, 344, 358,41, 44, 46, 48, 78, 376, 379, 80, 100, 101, 110, 111, 387, 112, 394,114, 116, 117, 121, 395, 133, 135, 137, 426, 174, 178, 186, 194, 196,202, 210, 213, 214, 217, 223, 227, 228, 233, 254, 271, 272 or 288 arehomologous to the peptides derived from other molecules, which are knownto sensitize human immune system.

To exclude this possibility, homology analysis was performed with thepeptide sequences as queries using BLAST algorithm(http://www.ncbi.nlm.nih.gov/blast/blast.cgi). No significant sequencehomology was revealed.

These results suggest that the sequences of SEQ ID NO: 19, 22, 30, 34,344, 358, 41, 44, 46, 48, 78, 376, 379, 80, 100, 101, 110, 111, 387,112, 394, 114, 116, 117, 121, 395, 133, 135, 137, 426, 174, 178, 186,194, 196, 202, 210, 213, 214, 217, 223, 227, 228, 233, 254, 271, 272 or288 are unique and thus possess a low risk of raising unintendedimmunologic response to any unrelated molecule.

Example 2 Materials and Methods Cell Lines

H2 (HLA-A02), human B-lymphoblastoid cell line, and COS7 were purchasedfrom ATCC.

Candidate Selection of Peptides Derived from INHBB

9-mer and 10-mer peptides derived from INHBB that bind to HLA-A*0201molecules were predicted using binding prediction software “BIMAS”(www-bimas.cit.nih.gov/molbio/hla_bind), which algorithms had beendescribed by Parker K C et al. (J Immunol 1994, 152(1): 163-75) andKuzushima K et al. (Blood 2001, 98(6): 1872-81). These peptides weresynthesized by Sigma (Sapporo, Japan) or Biosynthesis Inc. (Lewisville,Tex.) according to a standard solid phase synthesis method and purifiedby reversed phase high performance liquid chromatography (HPLC). Thepurity (>90%) and the identity of the peptides were determined byanalytical HPLC and mass spectrometry analysis, respectively. Peptideswere dissolved in dimethylsulfoxide (DMSO) at 20 mg/ml and stored at −80degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as antigen-presentingcells (APCs) to induce cytotoxic T lymphocyte (CTL) responses againstpeptides presented on human leukocyte antigen (HLA). DCs were generatedin vitro as described elsewhere (Nakahara S et al., Cancer Res 2003 Jul.15, 63(14): 4112-8). Specifically, peripheral blood mononuclear cells(PBMCs) isolated from a normal volunteer (HLA-A*0201 positive) byFicoll-Plaque (Pharmacia) solution were separated by adherence to aplastic tissue culture dish (Becton Dickinson) so as to enrich them asthe monocyte fraction. The monocyte-enriched population was cultured inthe presence of 1000 U/ml of granulocyte-macrophage colony-stimulatingfactor (GM-CSF) (R&D System) and 1000 U/ml of interleukin (IL)-4 (R&DSystem) in AIM-V Medium (Invitrogen) containing 2% heat-inactivatedautologous serum (AS). After 7 days of culture, the cytokine-induced DCswere pulsed with 20 mcg/ml of each of the synthesized peptides in thepresence of 3 mcg/ml of beta2-microglobulin for 3 hr at 37 degrees C. inAIM-V Medium. The generated cells appeared to express DC-associatedmolecules, such as CD80, CD83, CD86 and HLA class II, on their cellsurfaces (data not shown). These peptide-pulsed DCs were theninactivated by Mitomycin C (MMC) (30 mcg/ml for 30 min) and mixed at a1:20 ratio with autologous CD8+ T cells, obtained by positive selectionwith CD8 Positive Isolation Kit (Dynal). These cultures were set up in48-well plates (Corning); each well contained 1.5×10⁴ peptide-pulsedDCs, 3×10⁵ CD8+ T cells and 10 ng/ml of IL-7 (R&D System) in 0.5 ml ofAIM-V/2% AS medium. Three days later, these cultures were supplementedwith IL-2 (CHIRON) to a final concentration of 20 IU/ml. On day 7 and14, the T cells were further stimulated with the autologouspeptide-pulsed DCs. The DCs were prepared each time by the same waydescribed above. CTL was tested against peptide-pulsed T2 cells afterthe 3rd round of peptide stimulation on day 21 (Tanaka H et al., Br JCancer 2001 Jan. 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001 Apr.20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec. 15,10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9;Watanabe T et al., Cancer Sci 2005 August, 96(8): 498-506).

CTL Expansion Procedure

CTLs were expanded in culture using the method similar to the onedescribed by Riddell et al. (Walter E A et al., N Engl J Med 1995 Oct.19, 333(16): 1038-44; Riddell S R et al., Nat Med 1996 Feb., 2(2):216-23). A total of 5×10⁴ CTLs were suspended in 25 ml of AIM-V/5% ASmedium with 2 kinds of human B-lymphoblastoid cell lines, inactivated byMMC, in the presence of 40 ng/ml of anti-CD3 monoclonal antibody(Pharmingen). One day after initiating the cultures, 120 IU/ml of IL-2were added to the cultures. The cultures were fed with fresh AIM-V/5% ASmedium containing 30 IU/ml of IL-2 on days 5, 8 and 11 (Tanaka H et al.,Br J Cancer 2001 Jan. 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001Apr. 20, 84(8): 1052-7; Uchida N et. al., Clin Cancer Res 2004 Dec. 15,10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9;Watanabe T et al., Cancer Sci 2005 August, 96(8): 498-506).

Specific CTL Activity

To examine specific CTL activity, interferon (IFN)-gamma enzyme-linkedimmunospot (ELISPOT) assay and IFN-gamma enzyme-linked immunosorbentassay (ELISA) were performed. Specifically, peptide-pulsed T2(1×10⁴/well) was prepared as stimulator cells. Cultured cells in 48wells were used as responder cells. IFN-gamma ELISPOT assay andIFN-gamma ELISA assay were performed under manufacture procedure.

Results

Stimulation of the T Cells Using the Predicted Peptides from INHBBRestricted with HLA-A0201 and Establishment for CTL Lines Stimulatedwith INHBB Derived Peptides

CTLs for those peptides derived from INHBB were generated according tothe protocols set forth in “Materials and Methods” section above.Resulting CTLs having detectable specific CTL activity, as determined byIFN-gamma ELISPOT assay, are shown in FIG. 10. INHBB-A02-9-213 (SEQ IDNO: 143), INHBB-A02-9-174 (SEQ ID NO: 147), INHBB-A02-9-257 (SEQ ID NO:148), INHBB-A02-9-313 (SEQ ID NO: 149), INHBB-A02-9-139 (SEQ ID NO:150), INHBB-A02-9-8 (SEQ ID NO: 152), INHBB-A02-9-250 (SEQ ID NO: 153),INHBB-A02-10-179 (SEQ ID NO: 154), INHBB-A02-10-237 (SEQ ID NO: 156),INHBB-A02-10-313 (SEQ ID NO: 160), INHBB-A02-10-173 (SEQ ID NO: 161),INHBB-A02-10-256 (SEQ ID NO: 162), INHBB-A02-10-162 (SEQ ID NO: 163) andINHBB-A02-10-85 (SEQ ID NO: 166) demonstrated potent IFN-gammaproduction as compared to the control by IFN-gamma ELISPOT assay.Furthermore, the cells in the positive well number #7 stimulated withSEQ ID NO: 147, were expanded and CTL line was established. The CTL linehaving higher specific CTL activity against the peptide-pulsed target ascompared to the activity against target without peptide pulse wasdetermined by IFN-gamma ELISA (FIG. 11). The results herein demonstratethat the CTL line demonstrated potent IFN-gamma production against thetarget cells pulsed with corresponding peptide as compared to targetcells without peptide pulse. In the context of the present invention,the peptides which could establish CTL line were selected as potent CTLstimulation peptide.

In conclusion, novel HLA-A02 epitope peptides derived from INHBB wereidentified and demonstrated to be applicable for cancer immunotherapy.

DISCUSSION

Identification of new TAAs, particularly those that induce potent andspecific anti-tumor immune responses, warrants further development ofthe clinical application of peptide vaccination strategies in varioustypes of cancer (Boon T. et al., (1996) J Exp Med 183: 725-9.; van derBruggen P et al., (1991) Science 254: 1643-7.; Brichard V et al., (1993)J Exp Med 178: 489-95.; Kawakami Y et al., (1994) J Exp Med 180:347-52.; Shichijo S et al., (1998) J Exp Med 187:277-88.; Chen Y T etal., (1997) Proc. Natl. Acad. Sci. USA, 94: 1914-8.; Harris C C., (1996)J Natl Cancer Inst 88:1442-5.; Butterfield L H et al., (1999) Cancer Res59:3134-42.; Vissers J L et al., (1999) Cancer Res 59: 5554-9.; van derBurg S H et al., (1996) J. Immunol 156:3308-14.; Tanaka F et al., (1997)Cancer Res 57:4465-8.; Fujie T et al., (1999) Int J Cancer 80:169-72.;Kikuchi M et al., (1999) Int J Cancer 81: 459-66.; Oiso M et al., (1999)Int J Cancer 81:387-94.).

cDNA microarray technologies can disclose comprehensive profiles of geneexpression of malignant cells (Lin Y M, et al., Oncogene. 2002 Jun. 13;21:4120-8.; Kitahara O, et al., Cancer Res. 2001 May 1; 61:3544-9.;Suzuki C, et al., Cancer Res. 2003 Nov. 1; 63:7038-41.; Ashida S, CancerRes. 2004 Sep. 1; 64:5963-72.; Ochi K, et al., Int J Oncol. 2004 March;24(3):647-55.; Kaneta Y, et al., Int J Oncol. 2003 September;23:681-91.; Obama K, Hepatology. 2005 June; 41:1339-48.; Kato T, et al.,Cancer Res. 2005 July 1; 65:5638-46.; Kitahara 0, et al., Neoplasia.2002 July-August; 4:295-303.; Saito-Hisaminato A et al., DNA Res 2002,9: 35-45.) and, find utility in the identification of potential TAAs.Among the transcripts that are up-regulated in various cancers, novelhuman genes, termed CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTKand URLC10, were identified using these technologies.

As demonstrated above, CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2,TTK and URLC10, are over-expressed in various cancers but show minimalexpression in normal tissues. In addition, these genes have been shownto have a significant function related to cell proliferation. Thus,peptides derived from CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTKand URLC10 can serve as TAA epitopes, which, in turn, can be used toinduce significant and specific immune responses against cancer cells.

Thus, as CDH3, EPHA4, ECT2, HIG2, INHBB, KIF20A, KNTC2, TTK and URLC10are novel TAAs, vaccines using these epitope peptides find utility asimmunotherapeutics against various carcinomas or other diseaseexpressing these molecules.

INDUSTRIAL APPLICABILITY

The present invention identifies new TAAs, particularly those whichinduce potent and specific anti-tumor immune responses. Such TAAswarrants further development as peptide vaccines against diseasesassociated with the over-expression of CDH3, EPHA4, ECT2, HIG2, INHBB,KIF20A, KNTC2, TTK and/or, URLC10 e.g. cancers. All patents, patentapplications, and publications cited herein are incorporated byreference.

While the invention has been described in detail and with reference tospecific embodiments thereof, it is to be understood that the foregoingdescription is exemplary and explanatory in nature and is intended toillustrate the invention and its preferred embodiments. Through routineexperimentation, one skilled in the art will readily recognize thatvarious changes and modifications can be made therein without departingfrom the spirit and scope of the invention. Thus, the invention isintended to be defined not by the above description, but by thefollowing claims and their equivalents.

[Sequence list]ONC-A0704Psq.txt

What is claimed is:
 1. An isolated peptide of (a) or (b) below: (a) anisolated peptide of less than about 15 amino acids having cytotoxic Tcell inducibility, said peptide comprising the amino acid sequence ofSEQ ID NO: 80, 100 or 101; (b) an isolated peptide of less than about 15amino acids having cytotoxic T cell inducibility, wherein said peptidecomprises an amino acid sequence of SEQ ID NO: 80, 100 or 101 in which1, 2, or several amino acids are substituted, deleted, or added.
 2. Thepeptide of claim 1, wherein the peptide is selected from the groupconsisting of: (a) an isolated peptide consisting of the amino acidsequence of SEQ ID NO: 80, 100 or 101; and (b) an isolated peptidehaving cytotoxic T cell inducibility, wherein said peptide consists ofan amino acid sequence of SEQ ID NO: 80, 100 or 101 in which 1, 2, orseveral amino acids are substituted, deleted, or added.
 3. The peptideof claim 1, wherein the second amino acid from the N-terminus of theamino acid sequence of SEQ ID NO: 80, 100 or 101 is substituted withphenylalanine, tyrosine, methionine, or tryptophan.
 4. The peptide ofclaim 1, wherein the C-terminal amino acid of the amino acid sequence ofSEQ ID NO: 80, 100 or 101 is substituted with phenylalanine, leucine,isoleucine, tryptophan, or methionine.
 5. The peptide of claim 3,wherein the C-terminal amino acid of the amino acid sequence of SEQ IDNO: 80, 100 or 101 is substituted with phenylalanine, leucine,isoleucine, tryptophan, or methionine.
 6. A pharmaceutical compositionfor treating or preventing a disease associated with over-expression ofthe gene of SEQ ID NO: 5, said composition comprising a peptide of claim1 or a polynucleotide encoding the peptide.
 7. A pharmaceuticalcomposition of claim 6, wherein the disease associated with the gene ofSEQ ID NO: 5 is cancer.
 8. A pharmaceutical composition of claim 7,wherein the cancer is selected from the group consisting of bladdercancer, breast cancer, cervical cancer, cholangiocellular carcinoma,CML, colorectal cancer, endometriosis, esophageal cancer, gastriccancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renalcarcinoma, SCLC, soft tissue tumor and testicular tumor.
 9. A method ofinducing antigen-presenting cells having cytotoxic T cell inducibility,said method comprising the step of contacting an antigen-presenting cellwith a peptide of claim
 1. 10. A method of inducing cytotoxic T cells bycontacting a T cell with a peptide of claim
 1. 11. A method of inducingantigen-presenting cells having cytotoxic T cell inducibility, saidmethod comprising the step of transferring a gene comprising apolynucleotide encoding a peptide of claim 1 to an antigen-presentingcell.
 12. A method of inducing a cytotoxic T cell, said methodcomprising the steps of: (a) contacting an antigen-presenting cell witha peptide of claim 1, and (b) mixing the antigen-presenting cells ofstep (a) with a CD8+ T cell and co-culturing.
 13. A vaccine forinhibiting proliferation of a cell expressing the gene of SEQ ID NO: 5,wherein the vaccine comprises a peptide of claim 1 as an activeingredient.
 14. The vaccine of claim 13, wherein the cell expressing thegene of SEQ ID NO: 5 is a cancer cell.
 15. The vaccine of claim 14,wherein the cancer is selected from the group consisting of bladdercancer, breast cancer, cervical cancer, cholangiocellular carcinoma,CML, colorectal cancer, endometriosis, esophageal cancer, gastriccancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renalcarcinoma, SCLC, soft tissue tumor and testicular tumor.
 16. The vaccineof claim 15, formulated for administration to a subject whose HLAantigen is HLA-A24.
 17. A composition comprising a peptide of claim 1 ora polynucleotide encoding the peptide.
 18. A method of treating orpreventing a disease associated with over-expression of the gene of SEQID NO: 5 in a subject, said method comprising administering to saidsubject a vaccine comprising a peptide of claim 1, or a polynucleotideencoding said peptide.
 19. The method of claim 18, wherein the diseaseassociated with over-expression of the gene of SEQ ID NO: 5 is cancer.20. The method of claim 19, wherein the cancer is selected from thegroup consisting of bladder cancer, breast cancer, cervical cancer,cholangiocellular carcinoma, CML, colorectal cancer, endometriosis,esophageal cancer, gastric cancer, diffused type gastric cancer, livercancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreaticcancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor andtesticular tumor.